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FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY 

INVESTIGATION OF GAS-PHASE IONS 



By 

DAVID KAGE 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1999 






This dissertation is dedicated to the memory of my grandmother, Lila M. Brown, 
who would have laughed at all of this gobbledegook. 



ACKNOWLEDGMENTS 

There are many individuals whom I would like to thank that have contributed to my 
educational experience. Firstly, I would like to thank all of the previous and present 
members of the group. It was by working side-by-side with them, that most of this work was 
accomplished. The knowledge shared by all and passed through the ranks was invaluable. A 
big thank you goes out to Dr. Clifford H. Watson for always being there to answer basic 
questions. His knowledge of electronics, instrumentation, and trouble-shooting was a very 
big plus to have in the laboratory. I would like to thank Professor John R. Eyler for having 
the patience to let me stick around and try my best. 

My appreciation goes out to Professor Laszlo Prokai for the knowledge and assistance 
he gave me during the cyclodextrin project. Professor Martin Vala was very helpful on the 
polycyclic aromatic hydrocarbon project in more ways than one. Dr. Jan Szczepanski was 
extremely generous in helping me get the polycyclic aromatic hydrocarbon project off and 
going. His assistance with laser-oriented questions was always appreciated. I would like to 
thank the remaining members of my committee (Professors William Weltner, Robert 
Hanhrahan, and Lisa McElwee- White) for always having an open door if I needed to seek 
scientific advice or if I just wanted to discuss current events. 



in 



Often overlooked is the work performed by people behind the scenes. The work 
reported here is definitely no exception to this. Without the skilled talents of the machine 
shop and electronics shop, most of this work would not have been possible. The guys in the 
machine shop were always there to explain certain aspects of a design, fix parts when they 
broke, or help me redesign a critical piece on an instrument. The last members of this 
behind-the-scenes-group was Mrs. Lori Clark and all of the staff in the business office. Even 
when I would show up on a Friday at four o'clock with an emergency, they would still have a 
smile and the time to point me in the right direction for the money. 

I can't put into words how much I appreciate my family. I would like to tell my 
father, Jerry Kage, that I love him and that he can stop worrying about me because I am 
finally finished. I want to thank my mother, Marcia Kage, for always believing in me and for 
always putting up with me and all of my bitching for these past few years. I would like to 
thank my sisters, Dawn Hopping and Brenda Kage, for always being there when big brother 
needed to talk to someone. Finally, I would like to thank A. M. H. for giving me a reason to 
complete this venture. Her love, friendship, and patience through this endeavor were 
unparalleled. 



IV 



TABLE OF CONTENTS 

page 

ACKNOWLEDGMENTS iii 

LIST OF TABLES viii 

LIST OF FIGURES ix 

ABSTRACT xvi 

CHAPTERS 



1 . HISTORICAL INCEPTION AND FUNDAMENTAL PRINCIPLES 
OF FOURIER TRANSFORM ION CYCLOTRON RESONANCE 
MASSSPECTROMETRY 1 

Introduction 1 

Historical Overview 2 

The Basic Apparatus 5 

Motion of Trapped Ions 8 

CyclotronMotion 8 

TrappingMotion 10 

MagnetronMotion 11 

Experimental Procedure 12 

IonFormation 13 

IonExcitation 14 

ImpulseExcitation 17 

ChirpExcitation 17 

SWIFTExcitation 19 

IonDetection 19 

Conclusion 23 



2. GAS PHASE BINDING ENERGIES OF SELECTED HOST: 

GUESTCOMPLEXES 24 

Introduction 24 

CyclodextrinBackground 24 

Electrospraylonization 31 

Production of Charged Droplets 33 

Charged Droplet Shrinkage 35 

Mechanism of Gas-Phase Ion Production 36 

Collision-InducedDissociation 38 

Experimental 46 

Results 49 

a-CD:Tryptophan 50 

cc-CD:Proline 52 

a-CD:Lysine 52 

p-CD:Tryptophan 52 

P-CD:Histidine 53 

Discussion 53 



3 . MOTIVATION FOR INVESTIGATING POLYC YCLIC AROMATIC 

HYDROCARBONSOFASTROPHYSICALIMPORTANCE 71 

Introduction 71 

Background 71 

Related Studies 76 

CurrentEfforts 78 



4. PHOTODISSOCIATION AND ION-MOLECULE REACTIONS 

OFFLUORENECATIONS 81 

Introduction 81 

Background 81 

Experimental 82 

Results and Discussion 90 

Photodissociationvs. Irradiation Time 90 

Atomic vs. Molecular Hydrogen Loss 99 

Ion-MoleculeReactions 104 



VI 



5. PHOTODISSOCIATION AND ION-MOLECULE REACTIONS 
OF ACENAPHTHYLENE, DIPHENYLACETYLENE, AND 
NAPHTHALENECATIONS 110 

Introduction 110 

Acenaphthylene 110 

Diphenylacetylene 115 

Naphthalene 126 



6. CONCLUDING REMARKS 132 

Binding Energies for CD: Amino Acid Complexes 132 

Fluorene 133 

Acenaphthylene 135 

Diphenylacetylene 135 

Naphthalene 135 

LITERATURE CITED 137 

BIOGRAPHICAL SKETCH 150 



vii 



LIST OF TABLES 
Table page 

1 . A brief listing of selected physicochemical properties of the three most common 
cyclodextrin molecules. (Adapted from reference 101) 30 

2. The twenty essential amino acids along with their appropriate symbols and 

masses 51 

3. Conditions for studying the various [CD:amino acid]H + complexes 55 

4. Observed fragmentation channels and efficiencies for selected PAH cations using 
the ion-trap detector. (Reproduced from reference 197) 78 

5. A list of the twenty-four PAHs examined by Ekern et al. placed within the 
appropriate fragmentation category 79 



vni 






LIST OF FIGURES 
Figure Cage 

1 . Schematic representation of a typical cubic trapped analyzer cell commonly used 
in FT-ICR MS. The three pairs of parallel electrodes and their orientation with 

with respect to the magnetic field are depicted 7 

2. Origin of ion cyclotron motion. The path of an ion moving in the plane of the 
is bent into a circular orbit by the inward-directed Lorentz magnetic force 
produced by a magnetic field directed perpendicular to the plane of the paper. 
(Taken from reference 6) 9 

3. Schematic diagram of the natural motions of an ion trapped by a uniform magnetic 
and static electric field: co c (cyclotron), oo T (trapping), and © m (magnetron). 

The magnetron motion is circular about a guiding center that follows a contour 

of constant electric potential. (Adapted from reference 21) 12 

4. A general experimental pulse sequence that illustrates the four fundamental 

steps needed in order to obtain a mass spectrum using FT-ICR MS 13 

5. Incoherent ion cyclotron orbital motion (left) is converted to coherent (and therefore 
detectable) motion (right) by the application of an oscillating voltage to the 
excitation plates. Ions which are in resonance with the excitation frequency 

gain kinetic energy and spiral outward from the center of the cell into a larger 
cyclotron orbit (right). (Taken from reference 6) 15 

6. A Fourier excitation waveform and excitation spectrum for impulse (a) and chirp 

(b) excitation. (Taken from reference 7) 18 

7. An illustration of the principles of SWIFT excitation (a) and a SWIFT excitation 
depicting selective ejection of unwanted ions (b). (Taken from reference 7) 20 

8. A rotating monopole description of signal generation. Positive ions approach one 
plate, attracting electrons. As the ions continue moving in a circle, they 
approach the other plate and attract electrons. Thus, the ion motion induces a 
small AC (sine wave) current, an image current, in the detection plates. 

(Taken from reference 7) 21 



IX 



9. Overall depiction of an FT-ICR mass spectrometer. The upper diagram depicts 
the excitation of the ion packet by an externally applied alternating rf field. The 
lower picture shows the detection of the image current that is produced by the 
coherently orbiting ion packet on the two opposing detection plates to produce a 
time-domain signal. The time-domain signal is then converted to a voltage, 
digitized, and Fourier-transformed to yield a frequency-domain spectrum which 

is then converted to a mass spectrum. (Adapted from reference 54) 22 

10. Compounds 1-3 are the chemical structures of the three most common 
cyclodextrins: a-, p\ and y-cyclodextrin, respectively 26 

1 1 . Portion of a cyclodextrin molecule showing the glucose units connected through 
glycosidic a- 1,4 linkages 27 

12. Representation of the three most common cyclodextrins (a-, P-, and y-cyclodextrin) 
along with approximate dimensions and cavity volumes 28 

13. A simple depiction of the processes that occur in electrospray mass spectrometry. 
(Adapted from reference 115) 34 

14. Schematic representation of the ion evaporation model based on methanol as the 
solvent. The parent droplet that is created at the spray tip undergoes uneven 
fission as time passes. The depiction demonstrates how the parent droplet 
shrinks (losing about 2% of its mass) and loses charge (approximately 1 5%) as it 
produces daughter droplets while drifting towards the counter electrode 

(Adapted from reference 115) 37 

15. Resulting mass spectra following five stages of CID of FeS, + . (a) Isolation of Fe + 
following laser desorption and collisional cooling with argon and S 8 . (b) Reaction 
of Fe T with S g . (c) Isolation of FeS 10 + . (d) CID of FeS 10 + . (e) Isolation of FeS g + . 
(f)CIDofFeS 8 + . (g) Isolation of FeS 6 + . (h) CID of FeS 6 \ (i) Isolation of FeS 4 + . 
(j)CIDofFeS 4 \ (k) Isolation of FeS 2 + . (1) CID of FeS 2 + . (Taken from 

reference 139) 41 

16. Fourier transform ion cyclotron resonance mass spectrometer used to determine the 
gas-phase binding energies of cyclodextrimamino acid complexes. The instrument 
employed a shielded 4.7 T magnet, an external electrospray ionization source, 

and possessed three stages of differential pumping to achieve analyzer cell 

pressures on the order of 5.0 x 10" 9 Torr 47 



17. 


Typical pulse sequence used for the CID studies. HD is the Hexapole Dump, Q 
is the Quench pulse, IG is the Ion Generation pulse, MS is MS/MS Coarse 
Selection, IA is the Ion Activation pulse, E is the Excitation pulse, and D is the 








49 


18. 


Mass spectrum of the isolated [a-CD:Trp]H + at m/z 1 177. The unlabeled peaks 






demonstrate the inefficient ejection of unwanted ions during the isolation of the 








56 


19. 


The CID mass spectrum of [a-CD:Trp]H + showing the free protonated tryptophan 




20. 


at m/z 205 


57 


Percent fragmentation versus ion center-of-mass energy for [a-CD:Trp]H + . 




Extrapolation of this line to zero yields a threshold binding energy of 1 .32 eV 


58 


21. 


Mass spectrum of the isolated [cc-CD:Pro]H + at m/z 1088 


59 


22. 


The CID mass spectrum of [a-CD:Pro]H + showing the free protonated proline 




23. 


at m/z 1 16 


60 


Percent fragmentation versus ion center-of-mass energy for [a-CD:Pro]H + . 




Extrapolation of this line to zero yields a threshold binding energy of 1 .21 eV 


61 


24. 


Mass spectrum of the isolated [ct-CD:Lys]H + at m/z 1 1 19 


62 


25. 


The CID mass spectrum of [a-CD:Lys]H + showing the free protonated lysine 




26. 


at m/z 147 


63 


Percent fragmentation versus ion center-of-mass energy for [a-CD:Lys]H + . 




Extrapolation of this line to zero yields a threshold binding energy of 0.71 eV 


64 


27. 


Mass spectrum of the isolated [P-CD:Trp]H + at m/z 1339 


65 


28. 


The CID mass spectrum of [(3-CD:Trp]H + . The free protonated tryptophan at m/z 






147 can be seen in the expanded portion of the spectrum 


66 


29. 


Percent fragmentation versus ion center-of-mass energy for [P-CD:Trp]H + . 






Extrapolation of this line to zero yields a threshold binding energy of 0.58 eV 


67 


30. 


Mass spectrum of the isolated [p-CD:His]H + at m/z 1290 


68 


31. 


The CID mass spectrum of [P-CD:His]H + showing the free protonated histidine 






at m/z 156 


69 


xi 



32. Percent fragmentation versus ion center-of-mass energy for [P-CD:His]H + . 
Extrapolation of this line to zero yields a threshold binding energy of 0.73 eV 70 

33. Chemical structure and numbering system of the fluorene molecule (hydrogen 
atoms have been omitted from the structure) 82 

34. Results of DFT calculations 200 on the fluorene cation outlining the possible fragmen- 
tation pathways. The energies were calculated at the B3LYP/4-3 1 G level of theory 83 

35. Schematic representation of the 2 T instrument used to study the photodissociation 
of PAHs. (A) 2 T superconducting magnet, (B) ionization gauge, (C) inlet leak 
valves, (D) sample tubes, (E) gate valve, (F) oil diffusion pump, (G) solids probe 
port, (H) irradiation window, (I) connections for the analyzer cell and electron 

gun, (J) vacuum chamber 85 

36. Dimensions of the stainless steel (a) trapping tubes and (b) excitation and detection 
plates 86 

37. Analyzer cell that was used with the 2 T FT-ICR mass spectrometer to study 
PAHs. Depicted in the drawing are (a) the two stainless steel trapping tubes, (b) 
stainless steel tube cut into four equal segments used for the excitation and 
detection plates, and (c) virgin electrical grade Teflon (TFE) rings used to 
electrically isolate the different segments of the cell. The overall length of the 

cell is 12.791 5" and the diameter of the rings is 3.250" 87 

38. Depiction of the entire analyzer cell assembly used throughout the PAH studies. 
The analyzer cell was confined between four stainless steel rods that were held 
together by two stainless steel rings. The entire assembly was attached to a 
flange that contained the electrical feedthroughs that supplied voltages to the EI 

gun, trapping plates, and excitation plates 88 

39. A typical pulse sequence that illustrates the essential steps in obtaining a mass 
spectrum for the photodissociation studies of the fluorene cation 89 

40. A representative pulse sequence used to study the fragmentation as a function of 
irradiation time for the fluorene cation. The variable in these experiments was 
the length of the irradiation pulse (USER A). An ejection pulse was placed on the 
ion at m/z 167 (which was due to the presence of a carbon- 13 atom) in order not to 
complicate the spectra unnecessarily. This was done in order to ensure that 

the photodissociation products were derived from the parent ion at m/z 166 and 

not from the ion at m/z 167 91 



xn 



4 1 . Plot of fragmentation as a function of irradiation time from to 5000 ms for the 
fluorene cation 92 

42. Expanded portion of Figure 4 1 showing fragmentation as a function of irradiation 
time from to 1000 ms 93 

43. A typical mass spectrum of the fluorene cation. The daughter ions at m/z 163-165 
are a result of the EI process 94 

44. Mass spectrum of the fluorene cation after irradiation of 200 ms. Note that after 
only 200 ms the daughter ion at m/z 165 now dominates the mass spectrum 95 

45. Mass spectrum of the fluorene cation after irradiation for 500 ms. Note that after 
500 ms the dominant ion in the spectrum is the daughter ion at m/z 163 96 

46. Mass spectrum of the fluorene cation after irradiation for 5000 ms. Note that longer 
irradiation times lead to new ions as a result of ion-molecule reactions 98 

47. Pulse sequence used to determine if daughter ions of fluorene ions were formed by 
loss of atomic hydrogens or molecular hydrogens. In this experiment, the 

isolated parent ion at m/z 166 was exposed to the lamp for 1000 ms 100 

48. Pulse sequence used to determine if daughter ions of fluorene ions were formed by 
atomic hydrogen or molecular hydrogen loss. In this experiment, an ejection pulse 
was placed on the ion at m/z 1 64 during the irradiation event 101 

49. Mass spectrum of the fluorene cation after an irradiation of 1000 ms. In this 
experiment, the ejection pulse on the ion at m/z 164 was turned off, therefore, all 

the daughter ions from [M-Hp to [M-5H] + are visible in the spectrum 102 

50. Mass spectrum of the fluorene cation after an irradiation of 1000 ms. In this 
experiment, the ejection pulse on the ion at m/z 164 was turned on. The absence 
of the ion at m/z 163 proves that the hydrogens are being lost as atomic hydrogens 
and not as molecular hydrogens 103 

51. A typical mass spectrum obtained for the fluorene cation after longer irradiation 
times. This particular experiment had an irradiation time of 4000 ms. At 4000 ms, 
the parent ion has completely disappeared, leaving [M-3H] + as the dominant 
fragment ion. Also visible are ions (m/z 226-324) that result from ion-molecule 
reactions of the neutral parent and fragment ions 105 



xm 



52. A mass spectrum with identical conditions as the previous spectrum with on change; 
the lamp was turned off for this experiment (the 4000 ms was simply a delay time 
before detection). Note the absence of any ions between m/z 226-324 suggesting 
that these species are due to ion-molecule reactions initiated by the lamp 106 

53. A mass spectrum with identical conditions as in Figure 51 but with an irradiation 
time of only 500 ms. This spectrum is dominated by [M-3Hf with only a hint of 
any ion-molecule reactions occurring. This spectrum thus demonstrates that ion- 
molecule reactions are not important at shorter irradiation times 

(< 500 ms) 107 

54. Structure (a) results from a reaction between the neutral fluorene molecules and 
the fluorene fragment ions. The resulting ion of m/z 328 can further lose 
hydrogen atoms to form (b) which has a m/z of 324 109 

55. Chemical structure and numbering system for the acenaphthylene molecule (the 
hydrogen atoms have been omitted from the structure) 1 1 1 

56 A typical mass spectrum of the acenaphthylene cation after a 500 ms delay was 
placed before the detection pulse. The spectrum essentially depicts only the 
parent ion at m/z 152 112 

57. A mass spectrum of the acenaphthylene cation after a 60,000 ms delay time. As a 
result of the increased delay time, ion-molecule reactions are occurring that 
generate the ion near m/z 304 113 

58. Possible mechanism for the formation of the ion at m/z 304 resulting from an ion- 
molecule reaction between a neutral acenaphthylene and an acenaphthylene cation 1 14 

59. Mass spectrum of the acenaphthylene cation after irradiation for 60,000 ms by a 
xenon arc lamp. The spectrum depicts the generation of a new ion at m/z 228 
which likely forms as a result of the photodissociation of the ion at m/z 304 116 

60. Proposed scheme for the ion observed at m/z 228. This ion is a 
photodissociation product derived from the ion at m/z 304 11 7 

61. Chemical structure of the diphenylacetylene molecule 118 

62. A typical mass spectrum of the diphenylacetylene cation with a 500 ms delay 
placed before the detection event. At short delay times the mass spectrum is 
dominated by the parent ion at m/z 178 119 



xiv 



63 . A mass spectrum of the diphenylacetylene cation with a 5000 ms delay placed 
before the detect event 120 

64. Plot of percent abundance vs. delay time (lamp off) for the diphenylacetylene 
cation. At longer delay times, ion-molecule reactions resulted in a decrease in the 
abundance of the parent ion at m/z 178 along with an increase in the abundance 

of the product ion at m/z 356 121 

65. Two possible structures for the ion at m/z 356 that results from an ion-molecule 
reaction involving a neutral diphenylacetylene and a diphenylacetylene cation 1 22 

66. A typical mass spectrum of the diphenylacetylene cation after an irradiation of 
500 ms from a xenon arc lamp. The mass spectrum is dominated by the parent 
ion at m/z 178. The spectrum also depicts a significant abundance of the 

daughter ion at m/z 152 124 

67. A mass spectrum of the diphenylacetylene cation after an irradiation of 5000 ms. 
The spectrum depicts the formation of several new peaks which are most likely 
photofragments resulting from the photodissociation of the ion at m/z 356 125 

68. Two possible routes for naphthalene cation photodestruction. (Taken from 
reference 198) 127 

69. A typical mass spectrum of the naphthalene cation. The spectrum illustrates the 
tendency of the naphthalene cation to dissociate completely. The ions at m/z 102 
and 76 result from the EI process itself. 128 

70. A mass spectrum of the naphthalene cation after an irradiation pulse of 5000 ms. 
Note the formation of several new ions at masses above 128 129 

71 . Possible structures for the ions observed at m/z 202 and 250 resulting from 
irradiation of the naphthalene cation 130 



xv 



Abstract of Dissertation Presented to the Graduate 

School of the University of Florida in Partial Fulfillment 

of the Requirements for the Degree of Doctor of Philosophy 

FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY 

INVESTIGATION OF GAS-PHASE IONS 

By 

David Kage 

December 1999 

Chairman: Dr. John R. Eyler 
Major Department: Chemistry 

Fourier transform ion cyclotron mass spectrometry (FT-ICR MS) has received 
considerable attention for its ability to make mass measurements with a combination of 
resolution and accuracy that is higher than any other mass spectrometer. It can be used to 
obtain high-resolution mass spectra from ions generated by practically every known 
ionization method, to perform tandem mass spectrometric measurements, and to examine ion 
chemistry and photochemistry. Its versatility follows from the fact that it is an ion trapping 
instrument. The instrument mass analyzes and detects ions using methods which are unique 
among mass spectrometers. 

FT-ICR MS was used to measure the binding energies of cyclodextrimamino acid 
complexes in the gas-phase. Cyclodextrins are cyclic oligosaccharides that form truncated, 
cone-shaped molecules. The most common are referred to as a-, P-, and 



xvi 



y-cyclodextrin, which contain 6, 7, and 8 glucose units in the ring, respectively. The cavity 
that is formed by these molecules is hydrophobic, which lends to their ability as a "host" 
molecule for the study of host-guest chemistry. Collision-induced dissociation was used to 
measure the binding energies between the cyclodextrin host molecules and amino acid guest 
ions. 

FT-ICR MS was also used to study the photodissociation of polycyclic aromatic 
hydrocarbon cations that are of interstellar importance. It has become a widely accepted 
notion that PAH cations are the carriers of the diffuse interstellar bands that have been 
measured for years by astronomers. The unknown link is to find exactly what PAH cations 
are responsible for the bands. Experiments have been performed to answer this question. 
Two major themes are repeated for the fluorene, acenaphthylene, diphenylacetylene, and 
naphthalene cations: monitoring the generation of new ions formed as a function of trapping 
time in the analyzer cell, and analyzing the products that are generated after irradiation from 
a xenon arc lamp. Results from the first series of experiments reveal that new ions are being 
formed which result from ion-molecule reactions between neutral parents and parent ions. 
The second set of experiments show that the ions generated from the ion-molecule reactions 
are fragmenting into smaller ions as a result of the irradiation from the xenon arc lamp. 



xvii 



CHAPTER 1 

HISTORICAL INCEPTION AND FUNDAMENTAL PRINCIPLES 

OF FOURIER TRANSFORM ION CYCLOTRON 

RESONANCE MASS SPECTROMETRY 



Introduction 
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has, 
in recent years, developed into a very powerful analytical technique after years of 
promising anticipation. Since 1985, this technique has been the subject of four journal 
special issues, 1 " 4 three books, 5 " 7 and more than 60 review articles. 8 As of 1998 there were 
over 235 FT-ICR mass spectrometers located in several countries around the world that 
are being used to solve problems by a wide variety of scientists from analytical and 
physical chemists in academic settings to drug discovery scientists in the pharmaceutical 
community. In a relatively short time, FT-ICR MS has established itself as a powerful 
mass spectrometric technique that combines the advantages of ultra-high mass resolution 
and mass accuracy, is capable of utilizing a wide variety of ionization techniques, and can 
use a wide range of methods for structure characterization of the primary sample ions. 
With future improvements in magnet technology and with cheaper and more powerful 
computers, this technique will surely become the method of choice for mass spectrometry 
in the future. 



Historical Overview 
Fourier transform ion cyclotron resonance mass spectrometry is a mass spectrom- 
etric technique, whose beginnings can be traced back to conventional ICR mass 
spectrometry and Fourier transform nuclear magnetic resonance (FT-NMR) spectroscopy. 
The fundamental principles underlying ICR mass spectrometry were explained in 1930 by 
Ernest O. Lawrence 9 who invented the cyclotron particle accelerator (he was later 
awarded the Nobel Prize in physics in 1939 for this development). In 1932 Lawrence 
first demonstrated that a charged particle moving perpendicular to a uniform magnetic 
field is constrained to a circular orbit. 10 An orbit in which the angular frequency of the 
particle's motion is independent of the particle's orbital radius is characterized by the so- 
called cyclotron equation 

co = qBlm (1) 

where co is the angular frequency, q is the charge on the particle, B is the magnetic field 
strength, and m is the mass of the particle. Lawrence demonstrated that the cyclotron 
motion of a particle could be excited to a larger orbital radius by applying a transverse 
alternating electric field whose frequency matched the cyclotron frequency of the particle. 
This was a very significant discovery in that he demonstrated that a particle could be 
excited to a very large kinetic energy by use of small electric fields. The cyclotron 
particle accelerator has been a tremendous research tool used in the field of nuclear 
physics. 



3 
The first use in an analytical sense of the mass selective characteristics of the 
cyclotron motion of ions was the development of the Omegatron at the National Bureau 
of Standards by Sommer et al. in 1949."" 13 Their instrument used the frequency-selective 
cyclotron acceleration of ions by a radio frequency (RF) field, into an electrometer 
collector that detected the current produced by the ions that were ejected from a cell. 
Their instrument was designed to achieve the common objectives of early mass 
spectrometer development: high mass resolving power and high abundance sensitivity. 
They obtained the objectives, but due to stringent stability requirements for the 
electronics and for the need of a very high vacuum, the instrument was never 
commercially produced as a general purpose mass spectrometer. It did find service as an 
affordable analyzer for leak detection. 

The modern-day instrument can trace its direct ancestry back to the ICR 
spectrometer that was constructed in the mid-1960s in a collaborative effort between John 
Baldeschwieler's laboratory at Stanford University and a group of scientists at Varian 
Associates led by Peter Llewellyn. 14 The ICR technique soon became widely recognized 
as a preferred tool in the novel field of gas-phase ion chemistry with several instruments 
installed for basic research. These early instruments did have their share of disadvan- 
tages, primarily slow scan speeds and low mass resolution. With these limitations, it is 
obvious why the 1 970s did not see the advent of a commercially available instrument. 
Even so, the limited number of instruments opened up new areas for researchers; they 
provided an avenue to study ion-molecule chemistry, ion thermochemistry, and ion 
spectroscopy unavailable to them previously. 



4 
Possibly the most important period of evolution for the technique was initiated by 
professors Alan Marshall and Melvin Comisarow when they began to apply FT methods 
(which were universally accepted in the field of NMR spectroscopy) to the handling of 
ICR data. But before they could be successful, several key technological problems had to 
be mastered. The major setbacks included: 1) lack of a suitable means of storing a range 
of ions of widely varying mass-to-charge ratio (m/z) simultaneously for the length of time 
needed to perform a mass measurement by the FT method, 2) lack of a feasible method to 
allow resonant excitation of all ions over a broad m/z range (broadband excitation), 3) an 
acceptable method to detect all ions simultaneously, and 4) lack of a fast digitizer with 
sufficient memory and bit resolution available at that time. The full advantages of FT- 
ICR MS would not be appreciated until these major obstacles were overcome. 

Mclver solved the problem of ion storage with the development of the trapped ion 
cell in 1970. 15 Comisarow was able to solve the problem of broadband excitation by 
applying a rapid frequency sweep of a few tens of volts amplitude. This would be 
capable of exciting a wide mass range of ions, but in a much shorter time (as compared to 
a slow frequency sweep with an amplitude of a few millivolts used to excite one ion at a 
time over a long period). To detect all the ions simultaneously, Marshall and Comisarow 
decided to measure the image current induced in a set of nearby electrodes by the packet 
of ions undergoing cyclotron motion. With most of the technological obstacles solved, 
Marshall and Comisarow demonstrated the advantages of the FT mode of operation of 
ICR mass spectrometry in 1973. 16 " 18 The advantages of speed, high resolution, and 
effective computer data processing that accompanied the advent of FT techniques made 



5 
the instrument much more attractive as an analytical MS tool. After a few developmental 
years, Nicolet Instruments (now Finnigan) manufactured a commercial FT-ICR mass 
spectrometer in 1981 . Today, three companies market and sell FT-ICR mass spectrom- 
eters with the number of instruments used for studying the chemistry of gas-phase ions 
rising to over 235. 

The Basic Apparatus 

An FT-ICR instrument is a mass spectrometer, in other words, an instrument that 
observes the abundance of ions, resolved according to their masses. Most mass 
spectrometers operate on the basis of spatially separating the ions through a mass- 
dependent feature of their motion in a series of magnetic and/or electric fields while 
collecting the ions of different masses separately onto a detector. The FT-ICR approach 
is quite different since the ions are observed without separation or collection. The 
method is facilitated by using the absorption and emission of RF energy at the ion's 
characteristic (mass-dependent) cyclotron frequency as the ion undergoes cyclotron 
motion in a strong magnetic field. This fundamental detection principle (resonant 
absorption and emission of energy at a characteristic frequency) places this technique into 
the company of other resonance RF spectroscopies like NMR, electron paramagnetic 
resonance (EPR), microwave, and nuclear quadrupole resonance. 

All FT-ICR instruments have four main components in common. These are the 
need for a strong magnet, an analyzer cell, an ultra-high vacuum system, and a sophisti- 
cated data system. Only a short description of each will be given since each component 
in its own right could fill a chapter. The magnet can be either a permanent magnet, an 



6 
electromagnet, or more commonly, a superconducting magnet. The performance of the 
FT-ICR instrument improves as the magnetic field strength increases (discussed later). 
Superconducting magnets have field strengths that commonly range from 3 to 9.4 tesla. 
As magnet technology improves so does the field strength and with this comes an 
increase in the performance of FT-ICR MS. 

The second component is the analyzer cell. This is the heart of the instrument 
where ions are stored, mass analyzed, and detected. Several analyzer cell designs have 
been developed with specific tasks in mind, but the first, and possibly the most common 
design, is the cubic cell. It is composed of six plates arranged in the shape of a cube. 
The cell is situated in the heart of the magnetic field with one opposing pair of plates 
orthogonal and two pair of plates parallel to the magnetic field. The plates that are 
perpendicular to the magnetic field are referred to as the trapping plates. The two 
remaining pairs of plates are used to excite and detect the ions. Figure 1 depicts a basic 
cubic analyzer cell used in FT-ICR MS. A recent review presents the relative advantages 
of several analyzer cell designs. 19 

The third feature is the need for an ultra-high vacuum system. The performance 
of the FT-ICR instrument is more sensitive to pressure than other mass spectrometers. 
An ultra-high vacuum (pressures on the order of 10' 9 -10" 10 Torr) is required to achieve 
ultra-high resolution. To achieve these extremely low pressures, cryogenic or turbo- 
molecular pumps (backed by mechanical pumps) are typically preferred rather than oil 
diffusion pumps. 



V; 








B 



N 




Figure 1. Schematic representation of a typical cubic trapped analyzer cell commonly 
used in FT-ICR MS. The three pairs of parallel electrodes and their orientation with 
respect to the magnetic field are depicted. 



The final feature is the need for a very sophisticated data system. Some of the 
major components of the data station are a frequency synthesizer, delay pulse generator, 
broadband RF amplifier and pre-amplifier, a fast transient digitizer, and a powerful 
computer to coordinate all of the electronic devices during the acquisition of data, as well 
as to process and analyze the data. The FT-ICR technique has benefitted tremendously 
from the rapid growth and development of the semiconductor industry and will continue 
to benefit as new technological breakthroughs are made. 















8 
Motion of Trapped Ions 
Cyclotron Motion 

Ion cyclotron resonance spectrometers are based on the principle of cyclotron 
motion, by which the orbital movement of charged particles in an applied magnetic field 
can be described. How these charged particles are produced will be discussed later. In a 
strong magnetic field, a charged particle will experience an inwardly directed force 
known as the Lorentz force //that charged particle has some velocity component that is 
perpendicular to the direction of the field. In the absence of an electric field, the 
expression for the calculation of the Lorentz force experienced by an ion is given by 

F L = qv*B (2) 

where q is the charge of the ion, v is the ion's velocity, and B is the magnetic field 
strength. The cross product in eq 2 indicates that only those velocity components perpen- 
dicular to the magnetic field contribute to the Lorentz force. Figure 2 illustrates how the 
Lorentz force acts perpendicular to both the velocity and the magnetic field, resulting in a 
circular orbit of the charged particle. The centrifugal force for an object undergoing 
circular motion is given by 

F c = mJ/r (3) 



where m is the mass of the particle, v is the velocity of the particle, and r is the distance of 
the object from the center of rotation. 




® 



Figure 2. Origin of ion cyclotron motion. The path of an ion moving in the plane of the 
paper is bent into a circular orbit by the inward-directed Lorentz magnetic force produced 
by a magnetic field directed perpendicular to the plane of the paper. (Taken from 
reference 6) 



When the centrifugal force is equal to the Lorentz force, the ion achieves a stable 
circular orbit and Eqs 2 and 3 can be equated (in a simple treatment of the theory of ion 
motion) as demonstrated by 



mv/r = qB 



(4) 



The quantity v/r in Eq 4 is equal to the angular frequency, co, which is the number of 



10 
radians swept out by the ion per unit time. By substitution of this expression into Eq 4 
and rearranging the terms, the well-known cyclotron equation is obtained 

co c = qB/m (5) 

Angular frequency (radians per second) can be converted to linear frequency (cycles per 
second) by dividing by 2k. Finally, the celebrated cyclotron equation expressed in terms 
of SI units is given by 

v Q = qB/2nm (6) 

Eq 6 demonstrates that a group of ions with a given m/z always exhibit cyclotron motion 
at the same frequency v c (for a given value of B). This result is one of the reasons that 
FT-ICR is capable of measuring spectra with ultra-high mass resolving power. 
Additionally, the m/z of an ion is determined from its cyclotron frequency, and since 
frequency is the most accurately measured physical quantity, 20 FT-ICR provides a very 
accurate method for m/z determination. 
Trapping Motion 

Cyclotron motion is one of three natural motions an ion possesses as a result of 
being trapped by static magnetic and electric fields. A static magnetic field applied along 
the z-axis effectively confines ions in the x- and y-axes according to the cyclotron motion 
just described. However, ions are still free to escape in the z-axis, parallel to the 



11 

magnetic field. In order to mass analyze ions they must be trapped. Trapping of ions is 
accomplished by the use of a Penning ion trap, or as it is more commonly known in the 
FT-ICR MS community, an analyzer cell (described in the previous section). Trapping is 
generally accomplished by applying a small (~1 volt) electrostatic potential (same polarity 
as the ions of interest) to each of the two trapping electrodes (trapping plates). The ion 
trapping frequency has been previously derived. 6 The result is given by the expression 

co, = (laqV-/mtPf (7) 

where a is a constant that depends on the cell geometry, q is the charge of the ion, V T is 
the trapping voltage, m is the mass of the ion, and a is a characteristic trap dimension, 
which in the case of a cubic cell is the length of one side. In general, the trapping 
frequency is much smaller than the ICR orbital frequency (see Figure 3). 
Magnetron Motion 

The third natural motion is the "magnetron" motion which results from the 
relatively mass-independent precession of an ion along a path of constant electrostatic 
potential. Magnetron motion arises in a natural way as one of two solutions to the 
equations of (transverse) motion of an ion in static electric and magnetic fields. Although 
this motion can be excited, either intentionally or as an unintentional consequence of 
cyclotron excitation, it can be ignored in most FT-ICR applications. 



12 



Trapping Motion- 



Cyclotron Motion 



Magnetron Motion 




Figure 3. Schematic diagram of the natural motions of an ion trapped by a uniform 
magnetic and static electric field: co c (cyclotron), co T (trapping), and co m (magnetron). The 
magnetron motion is circular about a guiding center that follows a contour of constant 
electric potential. (Adapted from reference 21) 



Experimental Procedure 
The FT-ICR mass spectrometer operates in a very different fashion than most 
other types of mass spectrometers. With this technique, the principal functions of ion- 
ization, mass analysis, and ion detection occur in the same space (the analyzer cell) but 
are spread out in time, whereas with quadrupole and magnetic sector mass spectrometers, 
these events occur simultaneously and continuously in different parts of the mass spec- 
trometer. The basic series of events that occur in FT-ICR MS are referred to as a pulse 
sequence and consists of four events: quench, ion formation, ion excitation, and ion 
detection. This sequence of experimental events is depicted in Figure 4. The quench 



13 



event is used to empty the analyzer cell of any ions that may be present from a previous 
experiment. This is accomplished simply by applying antisymmetric voltages to the 
trapping plates. Under these conditions, ions are axially ejected (along the z-axis) from 
the cell in less than 1 ms (+10 and -10 V applied to the trapping plates). 

BASIC PULSE SEQUENCE 



Quench ionize 



Excite 



Detect 




time 



Figure 4. A general experimental pulse sequence that illustrates the four fundamental 
steps needed in order to obtain a mass spectrum using FT-ICR MS. 



Ion Formation 

Samples to be analyzed can be either a solid, liquid, or a gas. There are several 
possible techniques to get these samples into the gas. Solids probes are used to introduce 
solids of sufficient vapor pressure into the vacuum chamber, while leak valves and/or 
pulsed valves are used for liquids and gases. Once the samples are in the vacuum 
chamber, FT-ICR MS detects ions, therefore the samples need to be ionized. There are 
many different techniques that are used to ionize a sample depending on the specific 



14 
needs of the mass spectrometrist and the sample that is to be analyzed. These include (in 
no particular order) electron impact ionization (EI) 22 , chemical ionization (CI) 23 , laser 
desorption (LD) 24 " 27 , plasma desorption (PD) 28,29 , electrospray ionization (ESI) 30 " 34 , fast 
atom bombardment (FAB) 35 , secondary ion mass spectrometry (SIMS) 36 , field desorption 
(FD) 37 " 40 , and matrix-assisted laser desorption (MALDI). 41 " 43 Two of these ionization 
techniques (EI and ESI) will be discussed in more detail in later chapters. Appropriate 
references are cited for a more detailed discussion of each of the remaining techniques. 
Ion Excitation 

The ICR orbital motion that results from the magnetic and electric fields does not 
by itself generate an observable electrical signal. At its instant of formation the phase of 
each ion's orbital motion is random. In other words, an ion may start its cyclotron motion 
at any point along the circle depicted in the left diagram of Figure 5. Thus, any charge 
induced in either of the two opposed detector plates will be balanced, on the average, by 
an equal and opposite charge induced by an ion whose phase is 1 80° different. 

To circumvent the aforementioned problem of incoherency, the first step in FT- 
ICR MS detection is to excite the ions. There are several excellent references that discuss 
the fundamental process of excitation in FT-ICR. 44 " 46 In order to create a signal on the 
detector plates, an ion packet whose cyclotron orbits are initially centered on the z-axis 
must be made spatially coherent by moving the ion packet off-center. This is accom- 
plished by applying an oscillating resonant phase-coherent electric field excitation that 
accelerates the ions of interest into larger cyclotron orbits. An ion's orbital radius 



15 





Figure 5. Incoherent ion cyclotron orbital motion (left) is converted to coherent (and 
therefore detectable) motion (right) by the application of an oscillating voltage to the 
excitation plates. Ions which are in resonance with the excitation frequency gain kinetic 
energy and spiral outward from the center of the cell into a larger cyclotron orbit (right). 
(Taken from reference 6) 



can be determined from the following expression 



r = 1/qB (2mkT)'' 






(8) 






where q is the charge on the ion, B is the applied magnetic field strength, m is the mass of 
the ion, k is the Boltzmann constant, and T is the temperature of the ion. The previous 
equation can readily be derived from Eqs 4 and 9 which relates the translational energy of 
an ion to its temperature 



kT=mvJ/2 



(9) 



16 

As shown in Eq 8, the cyclotron radius of an ion can be increased by increasing its tem- 
perature (its kinetic energy). In addition to increasing an ion's cyclotron radius, excita- 
tion simultaneously achieves spatial coherency. The RF electric field component rotating 
in the same sense (in resonance with) as the ion of interest will push that ion continuously 
forward in its orbit. Thus, ions can be excited to detectable ICR orbital radii by a 
relatively small RF electric field. Therefore, all ions of a given m/z range can be excited 
to the same ICR orbital radius, by application of an RF electric field whose magnitude is 
constant with frequency. 

The goal for excitation is usually to excite all of the ions in the mass spectral 
range of interest to the same cyclotron orbital radius to produce a flat spectrum without 
mass discrimination. Several methods of ion excitation have been developed that achieve 
these conditions. The effects of an excitation waveform can be evaluated by displaying 
the excitation spectrum. This is obtained by performing an FT on the time-domain 
excitation waveform. The excitation spectrum portrays the amount of excitation at any 
frequency or mass, from which parameters such as ion radius, ion excitation energy for 
MS/MS, and overall evenness of the excitation can be observed. The three most common 
types of excitations are impulse excitation, 6 ' 4748 chirp excitation, 1745,49 and stored wave- 
form inverse Fourier transform (SWIFT) excitations. 50 All three of these excitation 
methods have been discussed in detail elsewhere and only a brief description of each will 
be given. 



17 
Impulse excitation 

An ideal delta function pulse (infinite amplitude, zero width) has a flat excitation 
spectrum and should excite all of the ions equally. In real world approaches to this idea a 
pulse of finite width and height is used (see Figure 6a). The shape of the pulse is not very 
important. The mass range of ions that are excited extends from infinite mass to a lower 
limit mass whose angular cyclotron frequency is of the order of co cmax = lit, where t is the 
pulse width. Mclver et al. 47 have discussed the quantitative aspects of impulse excitation 
and have shown that it is useful with a pulse amplifier delivering peak pulse amplitudes 
of the order of 1 kV. 48 
Chirp excitation 

The most commonly used excitation waveform is the "chirp", an RF pulse whose 
frequency sweeps rapidly over the range from the lowest to the highest (or highest to 
lowest) frequencies desired in the spectrum (see Figure 6b). A fairly flat excitation 
spectrum results if the frequency sweep traverses a constant number of Hertz per second. 
The mathematics of the FT with a chirp are slightly complicated, but were formulated 
long ago (see reference 6). The advantages of chirp excitation are its rather simple 
implementation and the ease with which a wide mass range can be excited without the 
need for large RF amplitudes and expensive amplifiers. Disadvantages are the somewhat 
nonuniform excitation of the ions, which becomes pronounced for ions near the edge of 
the swept frequency range (depicted in Figure 6b). 






18 



a) 



■S 



-> 



<-t 



time 




frequency 



b) 







Time 




FFT 

Power 
Spectrum 






Frequency 



Figure 6. A Fourier excitation waveform and excitation spectrum for impulse (a) and 
chirp (b) excitation. (Taken from reference 7) 



19 
SWIFT excitation 

In 1985, Marshall and coworkers introduced the SWIFT excitation method, which 
is the most satisfactory approach to achieving complete control over the excitation char- 
acteristics to date. 50 " 52 It is based on the fact that the excitation that is actually 
experienced by each ion, and therefore its final radius, is proportional to the amplitude of 
the excitation spectrum at its frequency. Recalling that the excitation spectrum is 
produced via an FT of the excitation waveform, a desired excitation waveform can be 
produced via an inverse FT of the excitation spectrum. The SWIFT technique specifies 
the excitation spectrum actually desired for the excitation waveform. This is ordinarily a 
square shape that extends the desired frequency range, as in Figure 7a. It can, however, 
just as well be a complicated shape with gaps, changing amplitudes, and other features as 
depicted in Figure 7b. The specified excitation spectrum is inverse Fourier-transformed 
to give the time-domain excitation waveform, as depicted in the right side of Figures 7a 
and 7b. If carried out precisely, this gives an excitation waveform that will excite each 
ion to exactly its preselected cyclotron radius. 
Ion Detection 

Detection in FT-ICR MS is based upon the principle of electric induction, 
whereby a current flows through a circuit in response to an accumulation of charge. The 
current will always flow in such a way that seeks to minimize the charge buildup. All 
ions of the same m/z are excited coherently and undergo cyclotron motion as a packet. 
As the orbiting ion packet passes the cell's electrodes (the detection plates), the coherent 
orbiting ion packet attracts electrons to first one and then the other of the two detection 



a) 



b) 




u 

3 



time 



.Transform, 




.Transform, 



time 



IM. 



frequency 



ejection limit 



frequency 



20 



Figure 7. An illustration of the principles of SWIFT excitation (a) and a SWIFT 
excitation depicting selective ejection of unwanted ions (b). (Taken from reference 7) 



plates through external circuitry (see Figure 8). This alternating current is referred to as 
the image current. 53 The periodic cyclotron motion of the ions produces a sinusoidal 
image signal which can be amplified, digitized, and stored for processing by a computer. 
The frequency of the detected sinusoid is nearly equal to the frequency of the cyclotron 
motion of the ions; it is exactly equal to the difference between the cyclotron and 
magnetron frequencies. 



21 



Electrons 



Receive 
Plates 




Electrons 



Figure 8. A rotating monopole description of signal generation. Positive ions approach 
one plate, attracting electrons. As the ions continue moving in a circle, they approach the 
other plate and attract electrons. Thus, the ion motion induces a small AC (sine wave) 
current, an image current, in the detection plates. (Taken from reference 7) 



Image current detection provides unique capabilities for FT-ICR MS. All other 
mass spectrometers detect ions by destructive collisions with an electron multiplier. 
Image current detection is non-destructive; the ions remain in the analyzer cell after the 
detection process has been completed. Since ion detection is nondestructive, ions can be 
repeatedly detected many times which improves the signal-to-noise ratio (S/N) since the 
ion signal increases as the square root of the number of detection events. Finally, the 
image current is converted to a voltage, amplified, digitized, and Fourier transformed to 



22 
yield a frequency spectrum that contains complete information about frequencies and 
abundances of all ions trapped in the cell. Finally, a mass spectrum can then be produced 
by converting frequency into mass (see Eq 6). Because frequency can be measured 
precisely, the mass of an ion can be determined to one part in 1 9 or better. 




Figure 9. Overall depiction of an FT-ICR mass spectrometer. The upper diagram depicts 
the excitation of the ion packet by an externally applied alternating RF field. The lower 
picture shows the detection of the image current that is produced by the coherently 
orbiting ion packet on the two opposing detection plates to produce a time-domain signal. 
The time-domain signal is then converted to a voltage, digitized, and Fourier-transformed 
to yield a frequency-domain spectrum which is then converted to a mass spectrum. 
(Adapted from reference 54) 



23 
Conclusion 
The purpose of this chapter was to provide the reader with a general summary of 
the FT-ICR technique, from its roots in the 1930s to the modern day instrument. Even 
after its relatively short existence (roughly some 25 years), FT-ICR MS has proven itself 
to be the method of choice for many researchers who are interested in the unique qualities 
this technique has to offer. The mass resolution and mass accuracy achieved by FT-ICR 
mass spectrometers is much higher than any other type of mass spectrometer. The tech- 
nology has come to a point where even pharmaceutical and biotechnology companies are 
now employing the use of FT-ICR mass spectrometers to assist them in their everyday 
analysis of samples. With future advancement of higher magnetic fields (at the National 
High Magnetic Field Laboratory and Battelle Pacific Northwest National Laboratory) as 
well as more powerful computers, improvements in not only mass resolution and mass 
accuracy but also in mass range, will undoubtedly make this technique the choice of mass 
spectrometrists in the future. 









CHAPTER 2 

GAS PHASE BINDING ENERGIES OF SELECTED 

HOST:GUEST COMPLEXES 



Introduction 

Gas phase binding energies of a series of amino acids trapped within the cavity of 
cyclodextrin molecules were measured using FT-ICR MS. To begin the chapter, a brief 
background regarding cyclodextrins will be offered. This is followed by a description of 
the experimental procedures that were applied; namely electrospray ionization and 
collision-induced dissociation. Finally, a discussion regarding the results obtained from 
these experiments along with a few comments on possible future experiments will be 
presented. 

Cyclodextrin Background 

Cyclodextrins (CDs) encompass a family of cyclic oligosaccharides that are 
produced from the enzymatic degradation of starch. The first reference to a substance 
that was later proven to be a CD was that of Villiers 55 in 1891. Villiers successfully 
isolated a white crystalline compound after digesting starch with the enzyme Bacillus 
amylobacter. It would be over ten years before a detailed report was published by 
Schardinger 56 that characterized the preparation and isolation of CDs. Schardinger was 
investigating strains of bacteria that were thought to be responsible for certain types of 
food poisoning that were occurring near the turn of the century. After digesting the starch 

24 



25 
with such a microorganism, he was able to isolate small amounts of two different 
crystalline compounds which appeared to be identical with the "cellulosines" reported by 
Villiers a decade earlier. Schardinger named the isolated microbe Bacillus macerans. 51 ' 
It was determined much later that, during the preparation process, the starch helix is 
hydrolyzed, and its ends are joined together through a- 1,4 linkages. 5960 The enzymes that 
are used in the digestion of starch are not specific as to the site of hydrolysis resulting in a 
product that exhibits a number of different cyclic and linear dextrins. 

Investigations into the chemistry of CDs have increased for several decades. 
Literature regarding structures, properties, and applications of CDs have been the subject 
of several books, 61 " 67 a number of review articles, 68 " 84 more than 800 patents, and countless 
papers in the years up to 1992. Their physical and chemical properties contribute to the 
broad interests from different scientific disciplines. CDs are the first and probably the 
most important examples of relatively simple organic compounds which exhibit complex 
formation with other organic molecules. They are excellent models of enzymes which led 
to their use as catalysts (for both enzymatic and nonenzymatic reactions), and they are 
natural products that are readily available to most researchers. 

The three most common CDs possess six, seven, and eight glucose units and are 
referred to as a-, 0-, and y-cyclodextrin, respectively. CDs that contain fewer than six 
glucose units are too strained to exist 85 whereas those that have more than eight are very 
soluble and difficult to isolate (though they have been identified by column chromatog- 
raphy). 86 The chemical structures of the three most common CDs are depicted in Figure 
10 while Figure 1 1 shows the glucose units in the relatively undistorted C, chair 



26 



CH20H 














t^> 



co* 



i 



s $ 




Vjp^ ? 



**• 



CHjOH 



^ 
<& 






^ 




3 




:A 



% 



Figure 10. Compounds 1-3 are the chemical structures of the three most common 
cyclodextrins: a-, p\ and y-cyclodextrin, respectively. 



27 







Figure 11. Portion of a cyclodextrin molecule showing the glucose units connected 
through glycosidic a- 1,4 linkages. 



conformation as well as the a- 1,4 linkages. This arrangement allows the CD to maintain 
an overall shape of a ring, or more accurately a conical cylinder, which is often described 
as a doughnut or wreath-shaped truncated cone. The wider side of the cone is created by 
the secondary 2- and 3-hydroxyl groups while the narrower side is created by the primary 
6-hydroxyl group (see Figure 10). The number of glucose units in the ring governs the 
overall dimensions of the cavity, as depicted in Figure 12. The cavity is lined with the 
hydrogen atoms and the glycosidic oxygen bridges. The nonbonding electron pairs of the 
glycosidic oxygen bridges are directed toward the interior of the cavity providing high 
electron density along with Lewis base characteristics. As a result of this arrangement of 
functional groups in the CD molecules, the cavity is relatively hydrophobic (compared to 
water) while the external faces are hydrophilic. Moreover, a ring of hydrogen bonds is 
also formed intramolecularly between the 2-hydroxyl and the 3-hydroxyl groups of 
adjacent glucose units. This hydrogen bonding ring gives the CD a remarkably rigid 
structure. 






28 



0.57 nm 




0.79 nm 



a-cyclodextrin (C 36 H 60 O 30 ), cavity volume 0.202 nnr 



0.78 nm 




0.79 nm 



p-cyclodextrin (C 42 H 70 O 35 ), cavity volume 0.377nm 



0.95 nm 




0.79 nm 



y-cyclodextrin (C 48 H 80 O 40 ), cavity volume 0.560 nm : 



Figure 12. Representation of the three most common cyclodextrins (a-, (3-, and y- 
cyclodextrin) along with approximate dimensions and cavity volumes. 






29 
It is the cavity that generates the attraction of many disciplines to the chemistry of 
CDs. As a result of the polar exterior and the relatively nonpolar interior, these com- 
pounds have been studied as "host" molecules for the inclusion of "guest" molecules 
which are capable of entering the cavity. The unique properties of the CD cavity explain 
some of the unusual features of these molecules; thus, they form inclusion complexes 
rather unspecifically with a wide variety of guest molecules. The only obvious require- 
ment for the guest molecule is that it must fit into the cavity, even if only partially. Based 
on this fact, it is not surprising to find that organometallics, 87 amino acids, 88 " 
peptides, 8891 " 93 aromatic molecules, 94 drugs, 9596 explosives, 97 and metal ions 98 are 
included, just to name a few in a long list of potential host species. 

The evolution of host-guest chemistry started in 1967 with the discovery of crown 
ethers by Pedersen. 99100 The term "host-guest chemistry" has been used to designate a 
variety of processes occurring in a number of research fields, such as organic, analytical, 
biological, pharmaceutical, and organometallic chemistry, and involving molecules and 
ions of different structures, dimensions, and properties. Restricting the definition of host- 
guest chemistry by considering the common elements that these disciplines possess is 
possible. In general, host-guest interactions involve the establishment of multiple non- 
covalent bonds between a large and geometrically concave organic molecule (the host) 
and a simpler organic or inorganic molecule or ion (the guest). Guest molecules or ions 
can be fully entrapped within the cavity or partially trapped as is the case with larger 
species such as peptides and proteins. Table 1 lists cavity dimensions as well as other 
properties of interest for the three most common CD molecules. 



30 



Table 1. A brief listing of selected physicochemical properties of the three most common 
cyclodextrin molecules. (Adapted from reference 101) 



cvclodextrin 



property . £t 



no. glucose units 



6 7 8 



empirical formula (anhydrous) C 36 H 60 O 30 C 42 H 70 O35 C 48 H 80 U 40 



mol. wt. (anhydrous) 

cavity length, A 

cavity diameter, A (approx.) 

ot D , deg. 

heat capacity (anhydrous solid), J mol"' K 

heat capacity (infinite dil'n.), J mol" 1 K"' 

pK a (25°C) 



972.85 1134.99 1297.14 

7.9 7.9 7.9 

-5.7 -7.8 -9.5 

+150.5 +162.0 +177.4 

1153 1342 1568 

1431 1783 2070 

12.33 12.20 12.08 



AH (ionization), kcal mol" 1 8.36 9.98 11.22 

AS (ionization), cal mol' K"' -28.3 -22.4 -17.6 

solubility (water, 25 °C), mol L" 1 0.1211 0.0163 0.168 

AH (solution), kcal mol"' 7.67 8.31 7.73 

AS (solution! cal mol"' K"' 13.8 a 112! HJ!_ 

a Mole fraction standard state. 



In aqueous solution, the slightly apolar CD cavity is occupied by water molecules, 
which is energetically unfavorable, and therefore, the cavity can be readily substituted by 
appropriate guest molecules which are less polar than water molecules. The dissolved 
CD is the host molecule with the driving force of complex formation being the substi- 
tution of the high-enthalpy water molecules by an appropriate guest molecule. Most 
frequently, the host:guest ratio is 1 : 1 ; this is the basis of "molecular encapsulation." A 
1 : 1 ratio is the most common case; however, 2:1,1 :2, 2:2, or even more complicated 
associations and higher order equilibria exist, almost always simultaneously. How these 
CD molecules are promoted into the gas-phase for analysis by mass spectrometry will be 
presented next. 



31 
F.lectros prav Ionization 
Recent advances in the biological sciences have generated a tremendous demand 
for the characterization of large biopolymers including peptides, proteins, oligonucleo- 
tides, and oligosaccharides. There has been tremendous pressure on the analytical 
chemistry community to keep up with these advances. A few of the daily challenges that 
are required of modern instrumentation are the need for rapid determination of molecular 
weight, purity, sequence, and site and nature of modifications. The biological sciences 
have greatly benefitted in recent years from improvements that have been made in mass 
spectrometry. More than ever, mass spectrometry has been called upon for the inves- 
tigation of biopolymers because it does not suffer from certain limitations of the classical 
techniques 102 such as gel electrophoresis or Edman degradation. The single event that 
finally demonstrated the usefulness of mass spectrometry for the biological sciences was 
the development of a new ionization technique. Historically, conventional mass spec- 
trometric methods could only be used on low molecular weight, volatile compounds. 
Larger species simply could not be promoted into the gas phase without undesirable 
degradation and/or fragmentation. Since the inception of electrospray ionization (ESI), 
the upper limit to molecular weight of proteins and biopolymers which can be studied has 
continued to increase to well over 100,000 kDa. 

The capability to impart multiple charges upon an analyte molecule is the 
principal feature of ESI that distinguishes it from other ionization techniques. These 
highly charged molecular ions, which normally exhibit little or no fragmentation, are 
reduced to a m/z range where conventional mass spectrometers routinely operate. The 



32 
combination of this fact along with the advantages of FT-ICR MS discussed previously, 
makes ESI FT-ICR MS quite possibly the most powerful analytical technique for studying 
very large proteins, biomolecules, or biopolymers. An example of the power of this 
coupling of techniques has been reported by Smith et al. m They recently reported the 
mass spectrum of a protein, bovine serum albumin (MW 66,430 Da), which showed ions 
with a charge distribution of +30 to +50 that corresponded to m/z values ranging from 
2214 to 1329, respectively. Electrospray ionization-mass spectrometry has been used to 
study a wide range of systems including proteins and glycoproteins, 104 nucleotides 
(including DNA, RNA, and oligonucleotides), 105 fullerenes, 106 synthetic polymers, 107 and 
inorganic transition metal complexes. 108 

Yamashita and Fenn 33109 were the first to demonstrate electrospray mass spec- 
trometry (ESMS) which was based upon the pioneering work of Dole et al. 30 The 
dramatic impact of this new technique was slow to be realized by most in the scientific 
community. 110 " 112 After several years of proven success and universal acceptance of the 
ESMS technique, much attention has now turned to understanding the mechanism of gas- 
phase ions production from solution phase analytes. There are at least three essential 
steps to consider: creation of charged droplets from dissolved electrolytes; solvent 
evaporation that leads to charged droplet shrinkage followed by repeated droplet disinte- 
grations (fissions); and the mechanism of gas-phase ion production from small, highly 
charged droplets. A brief description of the three processes is presented below. 



33 
Production of Charged Droplets 

The ES events are depicted in Figure 13. A voltage of 2-3 kV is applied to a 
small metal capillary (typically 0.2 mm o.d. and 0.1 mm i.d.) which is normally located 
from 1 to 3 cm from a larger planar counter electrode. The counter electrode will have an 
opening that leads to the mass spectrometric sampling system in most ESMS applications 
(for example, this opening allows ions to be transferred to the analyzer cell in FT-ICR 
MS). Due to the size of the capillary tip, the electric field in the atmosphere surrounding 
the tip is very high (E ~ 10 6 V m" 1 ). When the capillary of radius r c is located at a 
distance d from the planar counter electrode, the magnitude of E c for a given potential V c 
is given by" 3,114 

E c = 2VJrM^/r c ) (10) 

This equation provides the field at the capillary tip in the absence of solution. The 
electric field is proportional to the applied potential while the dominant geometric par- 
ameter is the capillary radius. 

Typical solutions used in ESMS will consist of a dipolar solvent in which electro- 
lytes are at least fairly soluble. Methanol (or methanol/water) is generally the solvent of 
choice with small amounts of acetic acid added as the source of electrolytes. For 
optimum operation of ESMS, low electrolyte concentrations ( 1 0" 6 - 1 0" 4 M range) are 
required. The choice of solvent, solvent mixtures, solvent mixture ratios, and 



34 



- 1-3 cm 



■a 
6 
S 
E 



Oxidation 






IoWq 





J 




i $ 



High voltage 
power supply 






Reduction 



Figure 13. A simple depiction of the processes that occur in electrospray mass 
spectrometry. (Adapted from reference 1 1 5) 



concentrations are, of course, parameters that can be different depending on the specific 
system of interest and which would need to be characterized for optimum efficiency. 

The applied electric field partially penetrates the liquid at the capillary tip. If the 
capillary is the positive electrode, the negative ions in the liquid will migrate toward the 
electrode while the positive ions migrate toward the liquid surface until the imposed field 
inside the liquid is essentially removed by this charge redistribution. Negative ions can 
also be generated for further investigation if the capillary is the negative electrode. The 



35 
accumulation of positive charge at the liquid surface tends to destabilize the liquid 
surface since the positive ions are repelled down field but cannot escape from the liquid. 
The surface is drawn out such that a liquid cone forms which has been referred to as a 
Taylor cone 116 in honor of Sir Geoffry Taylor. 

Eventually, as the electric field is increased above a certain value, the cone 
becomes unstable, resulting in a liquid filament with a diameter of a few micrometers 
(with a surface enriched with positive ions) being emitted from the cone tip. Separate 
droplets are formed downstream as the liquid filament becomes unstable with a continual 
increase of the electric field. The droplet surfaces are enriched with positive ions for 
which there are no negative counterions. The length of the unbroken liquid filament will 
decrease if the electric field is increased. 
Charged Droplet Shrinkage 

Since the initial size and the number of charges on the droplets depend on the 
spray conditions, it will be convenient for the next discussion to consider droplets that are 
formed by low flow rates (~5 uL min' 1 ) and concentrations that are less than 10" 3 M. 
Droplets formed by these conditions are considered to be monodisperse since they are 
small and have a narrow distribution of sizes. It has been shown that the size distribution 
peaks at a radius of about 1 .5 urn while possessing a charge on the order of 10" 14 C, which 
corresponds to approximately 50,000 singly charged ions. 117118 

The conditions that determine when the charge, Q, becomes sufficient to overcome 
the surface tension, y, that holds the droplet together are given by the Rayleigh equation" 9 



36 
^ R = 64^j< (11) 

where e is the permittivity of vacuum and R R is the Rayleigh radius. 

Larger droplets (within the micrometer range or larger) maintain their charge and 
do not emit gas-phase ions." 8120121 After the droplets have decreased in size to near the 
Rayleigh limit, they become unstable and begin to divide (undergo fission) into smaller 
droplets as seen in Figure 14. Studies have shown that the droplets do not produce 
offspring droplets of equal size and charge. 118120121 Furthermore, it was observed that the 
droplets tend to vibrate alternately from prolate to oblate shapes. These vibrations cause 
disruptions where the droplet releases a tail of much smaller offspring droplets. The 
offspring droplets take about 15% of the original charge and nearly 2% of the original 
mass with them when they are emitted. The radius of the offspring droplets is about one- 
tenth that of the parent droplets. 118122123 The total time for this sequence of events is in 
the hundreds of microseconds as seen from Figure 14 (calculated using methanol). 
Mechanism of Gas-Phase Ion Production 

Over the years, two different mechanisms have been proposed to account for the 
formation of gas-phase ions from the charged droplets. Dole et al. i0 devised the first 
mechanism which involved the formation of extremely small droplets ( R ~ 1 nm) that 
feature only one ion. Their mechanism allows gas-phase ions to evolve directly from 
these extremely small, solvent-evaporated droplets. How these extremely small droplets 
were formed or whether the process should include selectivity that may favor the 
formation of gas-phase ions A + relative to B + was not addressed in their proposal. 



37 



/V= 51250 
R = l.5 




N= 51250 
R = 0.945 



At = 462 us 




N= 43560 
J? = 0.939 




N = 43560 
« = 0.848 

74 MS 




Sf:g8 oooo 

20 droplets 



I 



yv= 37026 
R = 0.761 



JV= 37026 
R = 0.844 




70 ns 




W=326 OOOO 

« = 0.08 vw^ 



N = 31472 
fi = 0.756 



/V=278 OOOO 
R = 0.07 ^-"-"""^ 




39 ns 



N=2 



/V=278 
« = 0.03 

+■ O 



1 yV=236 
O R = 0.03 



R = 0.003 ' 



Figure 14. Schematic representation of the ion evaporation model based on methanol as 
the solvent. The parent droplet that is created at the spray tip undergoes uneven fission as 
time passes. The depiction demonstrates how the parent droplet shrinks (losing about 2% 
of its mass) and loses charge (approximately 15%) as it produces daughter droplets while 
drifting toward the counter electrode. (Adapted from reference 115) 



The second mechanism, proposed by Iribarne and Thomson, 124125 assumed that 
ion evaporation resulted from very small and highly charged droplets. Normally, the 
droplets have a radius of about 8 nm and roughly 70 elementary charges' 24125 when ion 
emission becomes competitive with Rayleigh fission. At this point, the droplet releases 
gas-phase ions rather than undergo fission to produce yet smaller droplets. As the 



38 
number of charges decrease, emission is still possible as a result of a decrease in the 
radius of the droplets by solvent evaporation. Thus, the Iribarne mechanism does not 
require the production of extremely small droplets that contain only one ion (as in Dole's 
theory). Iribarne emission can occur even when the droplet contains other solutes such as 
charge-paired electrolytes. At present, it is not possible to state with certainty which 
theory fits better with the available evidence. 

Collision-Induced Dissociation 

The goal of this project was to ascertain the gas-phase binding energies of a series 
of amino acids that were trapped within the cavity of CD molecules. The previous dis- 
cussion pertained to the generation of gas-phase ions; next a short discussion on how the 
binding energies were determined will be presented. 

Several different techniques have been developed for ion structure determination, 
but collision-induced dissociation (CID) remains one of the most useful and widely 
implemented mass spectrometric techniques, especially when employed in an MS/MS 
technique for complex mixture analysis. 126 " 128 Basically, this technique consists of 
isolating an ion of a specific mass, accelerating the chosen ion, and allowing it to pass 
through a collision gas. Upon collision, some of the ion's kinetic energy is converted into 
internal energy. This allows for a faster redistribution of energy throughout the normal 
modes allowing for higher energy fragmentations to occur. Normally, high kinetic 
energies (3-30 keV) are required to observe CID using mass-analyzed ion kinetic energy 
spectrometry (MIKES) in reverse-geometry mass spectrometers. 126 " 128 But Yost and 
Enke 129 were able to show that a low-energy (10-200 eV) CID process was possible with 



39 
high efficiency by using a triple-quadrupole mass spectrometer. This low energy pathway 
is readily accessible with an ICR spectrometer using the double-resonance technique to 
irradiate a given ion at its cyclotron frequency in order to accelerate it. The amount of 
kinetic energy transferred to the ion is limited to being less than that required to totally 
eject the ion from the cell, typically 10-1000 eV. As long as a collision gas is used at a 
sufficiently high pressure (~10" 5 Torr), dissociation may be observed instead of the 
ejection of the ion from the analyzer cell. Thus, their quadrupole results suggested that 
CID should be feasible in an FT-ICR mass spectrometer. In fact, CID was reported in the 
literature using conventional ICR mass spectrometers well before the quadrupole results 
appeared 130 " 133 but received little attention and remained essentially a curiosity. A 
possible reason for the lack of interest was due to the cumbersome nature of the 
experimental procedure for a conventional ICR mass spectrometer. 

The infinite parallel plate capacitor approximation 134135 (given in Eq 12) has 
commonly been used to calculate the translational energy imparted to an ion during the 
excitation stage of the FT-ICR CID process 

E^q'V^/Smd 2 (12) 

where q is the charge of the ion, Fis the amplitude of the RF excitation pulse, / is the RF 
pulse width, m is the mass of the ion, and d is the distance between the excitation plates 
of the analyzer cell. However, since the actual analyzer cell in many cases is a cubic cell, 
and therefore not an infinite parallel plate capacitor, the electric fields and translational 



40 
energies will undoubtedly be less than those values predicted for an infinite parallel plate 
capacitor. In fact, calculations and ion motion simulations 136 have demonstrated that 
excitation of ions located at the center of a cubic analyzer cell reached a radius that was 
only 72% of the theoretical radius that was calculated using an infinite parallel plate 
capacitor approximation. The actual excitation is even less since an ion's translational 
energy is proportional to the square of its radius. Therefore, actual excitation is only 0.52 
of that predicted by Eq 12. It is extremely important to keep the ion excitation time to a 
minimum. The important thing is that collisions between the ions and the collision gas 
occur after translational excitation, otherwise, uncertainties in the amount of energy 
actually imparted during the excitation process will result. 

Some of the most dramatic and promising MS/MS applications in FT-ICR MS 
involve sequential dissociations in which successive fragmentation of the parent ion into 
smaller and smaller fragments is followed by a series of excitation/observation steps on 
the successive fragments. 137138 An excellent illustration of MS" analysis was 
demonstrated by Freiser and Gord,' 39 where five CID steps were used, along with 
selective ejection, to proceed from FeS 10 + down to Fe + (see Figure 1 5). Carrying such 
multiple MS/MS observations to four or five steps is basically equivalent to a multisector 
or multi-quadrupole MS/MS experiment using a long (and impractical) series of sectors 
or quadrupoles. This experiment illustrated the use of an FT-ICR MS as a series of 
temporally separated mass analyzers. 

The basic CID process can be thought of in terms of two consecutive steps that 
occur on well-separated time scales. The first is a rapid step (~10"' 5 -10" 14 s) in which a 









41 








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44 
small amount of the initial translational energy of the accelerated ion is changed into 
internal energy of both the ion and target molecule (the target molecule also acquires 
translational energy). The next step in this process is the dissociation of the energized 
(and typically isolated) ion. The yield of product ions after collisional dissociation 
depends on the probability of unimolecular decomposition of the precursor ion after 
excitation. To explain the rates of such reactions, quasi-equilibrium theory (QET) has 
been used. 140 "' 44 In simple terms, the theory states that unimolecular decomposition 
reactions depend upon the random distribution of the internal energy of the ion among all 
the vibrational modes of that ion. In other words, the rate of decomposition is related to 
the probability of a given vibrational mode or modes acquiring enough energy to rupture 
bonds. 145 Since there are 3jV - 6 vibrational modes in a nonlinear ion that contains N 
atoms, the number of vibrational modes will be in direct proportion to the molecular mass 
for a given class of compounds. Because the random distribution of internal energy 
among the vibrational modes is required by QET, the average energy per mode must 
decrease with increasing molecular mass. Because the decrease is related to the inverse 
of the mass of the ion, the fragment ion yield should decrease similarly beyond some 
threshold. 

In the early days, low-energy CID mass spectra were observed in quadrupole 
reaction chambers of triple quadrupole or hybrid sector-quadrupole mass spectrometers, 
but in recent years low-energy CID has been performed more and more with FT-ICR 
mass spectrometers. Regardless of the choice of instrument configuration, an obvious 



45 
requirement is the presence of a collision cell that can be pressurized with a suitable 
target gas. 

Collisions that occur at large energies (keVs) are assumed to result in excitation of 
electronic internal modes (and to a lesser extent rotational-vibrational modes); collisions 
that occur at lower energies (<100 eV) will no longer result in efficient transfer of trans- 
lational energy to electronic internal modes. A typical interaction time of a selected ion 
of mass 200 and a translational energy of 30 eV with a target molecule over a few 
angstroms is on the order of 10" 13 s. This is longer than the time needed for internal 
electronic excitation and thus, the probability of such excitation is reduced with respect to 
excitation by high-energy (keV) CID. 146 

The interaction time of ca. 10' 13 s is comparable to the reciprocal of typical 
vibrational frequencies. Under these conditions, the collisions are nonadiabatic and the 
interaction is described as having an impulsive character that can effectively induce 
energy transfer. 146 The subsequent transfer of translational to vibrational energy is 
believed to occur by internuclear momentum transfer. 147 

In the so-called binary or spectator model, the selected ion and the target gas 
engage as essentially structureless elastic spheres. Momentum is transferred between the 
two bodies which leads to rotational-vibrational excitation of the ion, as well as the gas, 
along with a shift in momentum in the center of mass of each. If the ion is much larger 
than the collision gas, the gas will only interact with a small portion of the selected ion. 
The maximum amount of energy (center-of-mass kinetic energy, E com ) accessible for 



46 
internal excitation is given by Eq 13 148 

£com " £lab»VK + W t (Wp/m pl )] (13) 

where m p is the projectile mass, m pi is the impact portion of the projectile, and m x is the 
target mass. The elastic limit is thought to be reached when m p = m pi . 

With low-energy collisions, the composition of the target gas plays a much more 
important role than it does with high-energy collisions. The reason for this is that a 
different excitation mechanism (vibrational excitation) is at work. Furthermore, a larger 
portion of the maximum available energy is converted into internal energy of the target 
ion. Bursey and co-workers 149 " 151 demonstrated that heavier targets are preferred over 
lighter targets because they provide a larger E com . They found that collisions using helium 
transferred very small amounts of energy when compared with collisions using nitrogen, 
argon, or krypton. 149152 Specifically, for every volt change in E iab of the selected ion at a 
given pressure, there was an increase of 0.04, 0.25, and 0.32 eV, in maximum possible 
energy transferred when using helium, nitrogen, and argon, respectively. 152 

Experimental 

The experiments were conducted on a Bruker CMS 47X FT-ICR mass spec- 
trometer (Bruker Daltonics, Billerica, MA) incorporating a shielded 4.7 T supercon- 
ducting magnet (Magnex Scientific Limited, Abingdon, England) and a modified external 
electrospray ionization source (Analytica of Branford, Inc., Branford, MA). Figure 16 
depicts the instrument used throughout this work. The commercially-sold glass capillary 



47 



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48 
which utilized heated N 2 for desolvation was replaced in favor of a heated metal (brass) 
capillary (designed and built in-house) 153 which produced a more stable ion current. The 
capillary utilized a cartridge heater and under normal operating conditions was heated to 
between 100-130°C to assist in generating desolvated gas phase ions. 

The CDs (a-, (3-, and y-) and amino acids were provided by Dr. Lazslo Prokai and 
used without further purification. Typical solution concentrations were 10" 4 M for the 
CDs and 10" 3 M for the amino acids. The samples were sprayed from a water/methanol 
(50/50) solvent with a small amount of acetic acid added to provide charge. The 
solutions were introduced to the electrospray needle with the aid of a 74900 Series 
syringe pump (Cole-Parmer Instrument Company) normally operating at 60 ul hf '. The 
electrospray needle potential was normally maintained near +3500 V while the capillary 
was effectively kept at ground. The pressure in the external ion source was maintained at 
1 xlO" 6 Torr via pumping by an 800 L s" 1 cryopump (Edwards High Vacuum International, 
West Sussex, England). After entering the external ion source, the ions were guided to 
the analyzer region of the mass spectrometer by a series of electrostatic ion optics which 
were optimized for efficient ion transfer. Pressures in the analyzer region were typically 
maintained at 2x 10" 9 Torr by additional pumping from two 400 L s"' cryopumps. Immedi- 
ately before entering the analyzer cell, the ions were given a "sidekick" to minimize any 
z-axis loss. 154 Once in the cell, the ions were trapped using trapping potentials of +1.0 V 
and +1 .4 V on the two opposed trapping plates. 

Inside the analyzer cell, the ions were isolated using an RF notch ejection pulse 
(see Figure 17 for a typical pulse sequence). Following a 3 s cooling delay to allow for 



49 



HD 



Q IG 



MS IA E 



D 




Figure 17. Typical pulse sequence used for the CID studies. HD is the Hexapole Dump, 
Q is the Quench pulse, IG is the Ion Generation pulse, MS is MS/MS Coarse Selection, 
IA is the Ion Activation pulse, E is the Excitation pulse, and D is the Detection pulse. 









thermalization of the ions, the collision gas was introduced through either a piezoelectric 
pulsed valve (50 ms pulse) or a leak valve (Varian) to perform CID of the parent ions 
(cell pressure 1 xlO" 7 Torr). A 100 us RF activation was used to translationally excite the 
ions, which were then allowed to undergo collisions and fragment during a subsequent 
250 ms reaction delay. The resulting reactant and product ions were detected via 
frequency-chirp excitation. Broadband detection, covering a mass range of 50 to 2500 
amu, was utilized in these experiments. During detection, 64 spectra (64k data sets) were 
normally acquired and signal averaged in order to increase the S/N. 

Results 
The aim of this project was to determine the gas-phase binding energies between 
the three common CDs and the twenty essential amino acids, with thoughts of increasing 
the size of the guest molecule to include peptides and small proteins. The goals of this 
project were only partially realized. Results for a-CD with tryptophan, proline, and 



50 
lysine, and P-CD with tryptophan and histidine are presented below. A reference to the 
twenty essential amino acids is offered in Table 2 below. 
g-CD:Trvptophan 

The experimental parameters used to study the [a-CD:Trp]H + system (as well as 
the other systems) are summarized in Table 3 (found at the end of the chapter). The 
tryptophan side chain possesses a nitrogen-containing ring as well as a phenyl ring and is 
therefore classified as an aromatic amino acid. The cavity diameter of cc-CD has been 
reported to be between 4.7-5.7 A; therefore, the encapsulation and complexation of the 
side chain occurs easily by a number of interactions (hydrogen bonding, electrostatic, 
hydrophobic, and/or van der Waals). Figure 18 shows the isolated complex at m/z 1 177. 
A typical CID mass spectrum depicting the uncomplexed [Trp]H + (m/z 205) as well as 
other unidentified fragments is given in Figure 19. Fragmentation of this ion was 
measured as a function of the ion center-of-mass kinetic energy (an indirect measure of 
the amount of energy imparted to the ion in the collision). The variable throughout the 
experiment was the amplitude of the RF excitation pulse, which is to say, the amount of 
energy imparted in the collision. Attenuation of the RF excitation pulse varied from 1 1 to 
17 dB for the [a-CD:Trp]H + system (which corresponded to 240.2 to 89.8 Vp. p applied the 
analyzer cell plates). The threshold binding energy was determined by extrapolating the 
linear portion of the graph to zero (see Figure 20). The threshold binding energy for [cc- 
CD :Trp]H + was determined to be 1.32 eV. 







51 


Table 2. The twenty essential amino acids along with their appropriate symbols and 


masses. 






Hj 


M Oil 


-COOH 


(IN L-l 1 




R 




General Amino Acid Structural Unit With Distinctive R Group 


Name R group 


Svmbols 


Monoisotopic Mass Average Mass 


Nonpolar, aliphatic R groups 






Glycine -H 
Alanine -CH 3 
Proline -(CH 2 ) 3 - 
Valine -CH(CH 3 ) 2 
Leucine -CH 2 CH(CH 3 ) 2 
Isoleucine -CH(CH 3 )C 2 H 5 


Gly,G 
Ala, A 
Pro, P 
Val,V 
Leu, L 
He, I 


57.02146 57.0520 
71.03711 71.0788 
97.05276 97.1167 
99.06841 99.1326 
113.08406 113.1595 
113.1595 113.1595 


Aromatic R groups 






Phenylalanine --CH 2 C 6 H 5 
Tyrosine -CH 2 C 6 H 4 OH 
Tryptophan -CH 2 (C 2 H 2 N)C 6 H 4 


Phe, F 
Tyr,Y 
Trp,W 


147.06841 147.1766 
163.06333 163.1760 
186.07931 186.2133 


Polar, uncharged R groups 






Serine -CH 2 OH 
Threonine -CH(CH 3 )OH 
Cysteine -CH 2 SH 
Asparagine -CH 2 CONH 2 
Glutamine -(CH 2 ) 2 CONH, 
Methionine -(CH,) 2 SCH 3 


Ser, S 
Thr,T 
Cys,C 
Asn, N 
Gln,Q 
Met, M 


87.03203 87.0782 
101.04768 101.04768 
103.00919 103.1448 
114.04293 114.1039 
128.05858 128.1308 
131.04049 131.1986 


Positively charged R groups 






Lysine -(CH 2 ) 4 NH 2 
Histidine -CH 2 (C 3 H 3 N 2 ) 
Arginine -(CH 2 ) 3 NH-C(N 2 H 3 ) 


Lys,K 
His, H 
Arg,R 


128.09496 128.1742 
137.05891 137.1412 
156.10111 156.1876 


Negatively charged R groups 






Aspartate -CH 2 COOH 
Glntamate -(CH,),COOH 


Asp, D 
Glu. E 


115.02694 115.0886 
129.04259 129.1155 









52 
g-CD:Proline 

Proline has an aliphatic ring and is, therefore, classified as a nonpolar amino acid. 
Figure 21 is the mass spectrum of the isolated [a-CD:Pro]H + at m/z 1088. A typical CID 
mass spectrum depicting the free protonated proline (m/z 1 16) as well as the parent ion is 
presented in Figure 22. Attenuation of the RF excitation pulse used for the [a-CD:Pro]FT 
system ranged from 13 to 19 dB (169.7 to 65.2 VJ. Figure 23 highlights the linear 
portion of the graph which resulted in a threshold binding energy of 1.21 eV when 
extrapolated to zero. 
q-CD:Lysine 

The side chain of lysine possesses an amine group and lysine is thus classified as a 
positively charged amino acid. The isolated parent ion (m/z 1 1 19) can be seen in the 
mass spectrum shown in Figure 24. Figure 25 is a representative CID mass spectrum of 
[a-CD:Lys]H + showing the free protonated lysine (m/z 147) as well as the parent ion. 
Attenuation of the RF excitation pulse used for [a-CD:Lys]FT varied from 12 to 30 dB 
(204.9 to 17.83 V ). Figure 26 depicts the linear portion of the graph which resulted in a 
threshold binding energy of 0.71 eV when extrapolated to zero. 
p-CD:Trvptophan 

The cavity of the P-CD is larger than that of the a-CD; therefore, the encapsul- 
ation of the amino acids should be facilitated. The isolated [P-CD:Trp]H + at m/z 1339 
can be seen in the mass spectrum depicted in Figure 27. A typical CID mass spectrum is 
illustrated in Figure 28. The free protonated tryptophan (m/z 205) can be seen in the 
expanded region of the spectrum. Attenuation of the RF excitation pulse for 



53 
[p-CD:Trp]H + varied from 1 1 to 20 dB (240.2 to 57.1 V^). Figure 29 depicts the linear 
portion of the graph which resulted in a threshold binding energy of 0.58 eV when 
extrapolated to zero. 
ft-CD:Histidine 

Histidine is classified as a positively charged amino acid since the side chain 
contains a ring with two nitrogen atoms. Figure 30 shows the mass spectrum depicting 
the isolated parent ion at m/z 1290. Attenuation of the RF excitation pulse for [p- 
CD:His]H + ranged from 12 to 21 dB (204.9 to 50.7 V„). A CID mass spectrum showing 
the free protonated histidine (m/z 1 56) as well as the parent ion is given in Figure 3 1 . 
The threshold binding energy was determined to be 0.73 eV from the graph in Figure 32. 

Discussion 

Unfortunately, a great deal of information cannot be inferred from these data due 
to the limited number of systems. A comparison can be made between the complexation 
of tryptophan to a-CD and p-CD. The intermolecular forces that bind the tryptophan to 
the interior of the CD would presumably be stronger for the a-CD case due to the smaller 
diameter. With a smaller cavity diameter (closer proximity of atoms), the a-CD would 
bind more tightly to the tryptophan than the P-CD would. This is reflected in the two 
threshold binding energies that were measured; 1.32 and 0.58 eV for the a-CD and P-CD 
systems, respectively. It appears that twice the energy was necessary upon collision to 
eject the tryptophan from the a-CD cavity than the P-CD cavity. 



54 
The threshold binding energy between a-CD and lysine, tryptophan, and proline 
can be compared since the cavity size in each case remains the same. Given the size of 
the a-CD cavity the binding strength should be the greatest for tryptophan and the 
weakest for lysine. Since the side chain of lysine is simply a straight chain (weakest 
intermolecular forces), the lysine would not be encapsulated as tightly as with the other 
two amino acids. As for the tryptophan and proline case, they both possess a ring and, 
therefore, would be of comparable size. However, the tryptophan would extend deeper 
into the cavity. In addition, the intermolecular forces holding the tryptophan complex 
together would be stronger due to the increased mass over the proline complex (all other 
intermolecular forces taken to be equal). The calculated binding energies did, in fact, 
follow this trend yielding 1.32, 1.21, and 0.71 eV for a-CD with tryptophan, proline, and 
lysine, respectively. 

































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CHAPTER 3 

MOTIVATION FOR INVESTIGATING POLYCYCLIC AROMATIC 

HYDROCARBONS OF ASTROPHYSICAL IMPORTANCE 

Introduction 

The work presented in chapters four and five was undertaken with the purpose of 
studying the products of photodissociation from a series of polycyclic aromatic hydro- 
carbon cations that have potential interest within the astrophysical community. The goal 
was to find laboratory analogues of the species that are possible carriers of the diffuse 
interstellar bands that have been observed in specific regions of space. An appropriate 
background discussion will be offered in the present chapter to show the importance of 
these studies. Following the background discussion, a brief review will provide previous 
experimental results along with the current direction of our research. 

Background 

Polycyclic aromatic hydrocarbons (PAHs), more simply known as polyarenes, 
embody an extraordinarily large and diverse class of organic molecules that are generated 
from fused benzene rings. The major sources of PAHs on this planet are crude oil, coal, 
and oil shale. The fuels produced from these fossil sources constitute the primary source 
of energy for the industrial nations of the world, and the petrochemicals produced from 
these raw materials are the basis of the synthetic fibers and plastics industries. Major 
considerations of this family of molecule have been their prominent roles as 



71 



72 
environmental toxins, 155 exemplified by the discovery in the mid-1 930s of the carcin- 
ogenic properties of benzo[a]pyrene and other polyarenes. This discovery was an 
important landmark in biomedical science since it was the first indication of disease 
caused not by a microorganism, but by a relatively simple organic molecule. Since this 
discovery, researchers have found significant levels of these environmental contaminants 
in the air we breathe, the food we eat, and the water we drink; most of which can be 
attributed to the burning of fossil fuels as we have continued to exist as an industrialized 
nation. 

During the past two decades, PAHs have again received a considerable amount of 
interest, but this time it was the astrophysical community that found PAHs to be 
intriguing molecules. During this time period, researchers have presented insurmountable 
evidence that PAHs are also important members of the interstellar medium (ISM), quite 
possibly being the third most abundant detected molecules behind H 2 and CO. 156 Yet, the 
likely presence of PAH molecules and ions beyond the earth's atmosphere in space is still 
somewhat of a novelty for many chemists. Even though they have not been unequiv- 
ocally identified, aromatic and PAH molecules and ions are now generally accepted by 
the astrophysical community to be present in interstellar and circumstellar environments. 
Some have even argued for the presence of nonplanar PAHs and of hollow cages of 
carbon atoms known as fullerene molecules. 

The hypothesis that PAHs are present in interstellar and circumstellar environ- 
ments already has a substantial historical record. 157 " 162 Discussions dealing with the 
presence of PAHs in interstellar environments began with the visionary suggestion, 163 in 



73 
1956, that related carbonaceous species are responsible for visible diffuse absorption 
bands. This fact crystallized with the discovery 164 that some astronomical objects emit a 
broad infrared emission band which peaks at 3050 cm' 1 as well as other unique emission 
features 165166 which peak in the region between 1610 and 890 cm" 1 . Astronomical objects 
which emit these features include regions associated with individual stars such as H-II 
regions and reflection nebulae as well as interstellar clouds like the IR Cirrus, both in our 
own and other galaxies. 167 " 170 Soon after their discovery, these infrared emission features 
were attributed to infrared fluorescence from molecular-sized emitters excited by the 
absorption of ultraviolet and visible photons. 171172 The idea that the fluorescence 
originated from vibrations of chemical groups attached to aromatic constituents of 
amorphous carbon particles 173 led to the proposal that individual PAH molecules are 
responsible for the infrared emission due to their stability against photodissociation and 
the resemblance of laboratory infrared fluorescence data of such species to the 
astrophysical spectra. 174 " 176 PAH molecules have also been proposed as carriers of visible 
diffuse interstellar bands. 156177178 Presently, the PAHs responsible for the infrared 
features are thought to be more abundant (-17% of the cosmic carbon) than all of the 
other known gaseous interstellar organic molecules combined. 181 

Current models of interstellar and circumstellar chemistry have emphasized planar 
PAHs with arrangements of hexagonal rings. These rings are more or less compact 
("catacondensed") with the general formula C 6p 2 H 6p such as coronene (C 24 H 12 ) or 
elongated polyacenes with the general formula C 4n+2 H 2n+4 such as naphthalene (C, H g ), 
anthracene (C, 4 H 10 ), or tetracene (C lg Hi 2 ). 157 PAHs with loose arrangements of hexagonal 



74 
rings, such as those bound by single carbon-carbon bonds, have largely been neglected. 
In addition, non-hydrocarbon aromatic molecules have also been excluded. Neutral and 
positively-charged fused-ring molecules such as pyrene, coronene, and ovaline, either 
completely or partially hydrogenated, have been invoked to account for both the observed 
broad IR emission features in nebulae 174 " 176 and the observed diffuse interstellar 
absorption bands. 177 " 179 Recent detection of additional sharp emission features 180 has led 
to the proposal that much simpler linear fused-ring molecules such as naphthalene, 
anthracene, and tetracene are responsible for the infrared emission and that benzene may 
also be present in these environments. 181 For example, anthracene has been suggested as 
the most abundant of these linear polyacenes in the Orion Ridge. 181 

Diffuse interstellar bands (DIBs) are ubiquitous absorption features in astro- 

o 

nomical spectra. They absorb from approximately 4,400 A into the near infrared. 
Identifying the carriers of the DIBs has become a classic spectroscopic problem of the 
20 th century. Since their original discovery in 1922 by Heger, 182 these bands have 
challenged spectroscopists, astronomers, and physicists, and their origin remains the 
longest standing unsolved problem in all of spectroscopy. 183 During this time, so many 
suggestions have been made, experiments carried out, and theories proposed that it would 
be impossible to review them in this work. The experimental challenge was succinctly 
stated by Johnson 184 almost thirty years ago, "...one not only has to match 25 diffuse 
interstellar lines as far as wavelengths are concerned, but also as far as intensity. In 
addition, the... interstellar line widths vary from 40 A to 1 A... and are invariant to 0.1 
A..." Since that time, the number of DIBs has grown to nearly 160 and their relative 



75 
intensities have been shown to vary from one line-of-sight to another. In addition, some 
DIBs seem to associate in loosely connected families. 185 " 187 Wavelength invariance has 
been taken to indicate that the carriers cannot reside in or on dust particles. This is 
because particle size, shape, and composition influence peak position and profile, and it is 
difficult to imagine that the grains along all lines-of-sight are exactly the same. 

The criteria which must be met for a particular material to be considered for 
acceptance as a DIB carrier are that its visible and near-infrared absorption features match 
the known DIBs in wavelength, bandwidth, and relative intensities while not possessing 
additional features that are absent in the interstellar spectra. Of the aforementioned 160 

o 

DIBs, a carrier for two has almost certainly been identified; the DIBs at 9577 A and 
9632 A closely agree with expected spectral features due to the C 60 fullerene cation. 188 
Current theories from leading researchers in the field believe that the carriers are PAH 
cations or, to account for the discrepancies in peak intensities, mixtures of PAH cations 
and neutrals. 

Since PAHs are believed to be ubiquitous and abundant in the interstellar 
medium 175176 and are stable against ultraviolet photodissociation, they are attractive 
candidates for the DIB carriers. Moreover, a large fraction of the PAHs are expected to 
be ionized in the interstellar medium, 156176 " 178 and thus, absorb lower-energy photons 
(mainly in the visible and near-infrared regions of the spectrum) than their neutral 



|M 

precursors. 






76 
Related Studies 
The only available data on PAHs for many years have come from absorption 
spectra of neutral and ionized PAHs suspended in perturbing media (solid phase 189 191 or 
solution 192 ) or from gas-phase photoelectron spectra which do not provide information on 
all the possible optical transitions. 193 Allamandola was one of the first investigators to 
systematically measure the spectroscopic properties of neutral and ionized PAHs in the 
ultraviolet, visible, and near-infrared range (1,800-9,000 A) under conditions relevant to 
astrophysical environments. This was accomplished by studying the isolated species in 
the least-perturbing solid medium known, neon matrices at 4.2 K. Neon generally 
produces shifts in vibronic positions of only a few tenths of a percent with respect to the 
gas phase. 194 Since their first report on naphthalene (the smallest PAH) in 1991, the 
Allamandola laboratory has published several articles dealing with matrix-isolated PAH 
and PAH cations. But, as expressed by Allamandola, even at 4.2 K in a neon matrix, 
there still exist matrix effects that are absent in the interstellar medium. The ideal 
situation would be to study these systems in the gas phase where perturbations from a 
matrix are absent. 

Boissel and co-workers 195 reported the results of a study using a FT-ICR MS with 
the Penning trap placed in an ultrahigh vacuum cell attached to a closed cycle helium 
cryostat. The temperature of the parts ranged from 12 K to 30 K, ensuring a very low rate 
of ion-neutral collisions (confirmed by the long trapping times of up to ten minutes). Ions 
were produced by laser ablation of a solid PAH pellet located near one of the trapping 
plates. The trapped ions were irradiated by a focused cw xenon arc lamp that had a 



77 
computer-controlled mechanical shutter to allow light into the cell. The length of irradi- 
ation (in other words the amount of energy imparted to the ions) was varied throughout 
the experiments. Mass spectra recorded the fragmentation after irradiation of the anthra- 
cene, anthracene-d 10 , and pyrene cations. The photodissociation pathways were found to 
be the loss of C 2 H 2 , C 2 D 2 , and H 2 (or two hydrogen atoms) for the three previously listed 
ions, respectively. 

Two more papers appeared in the literature in late 1997 that pertained to PAHs 
and astrophysical implications. Ekern and co-workers 196 used FT-ICR MS to study the 
coronene and naphtho[2,3-a]pyrene cations. Upon irradiation from a xenon arc lamp, 
each of the two ions was found to dehydrogenate completely to leave the C 24 + bare carbon 
cluster cation. In the second paper, Wang et a/.' 97 examined the CID mass spectra of a 
series of PAH cations using a modified ion trap. Ions of interest were the naphthalene, 
acenaphthylene, acenaphthene, anthracene, phenanthrene, pyrene, coronene, and the 
corannulene cation. Mass spectra were recorded after the selected PAH cation was 
allowed to undergo collisions with argon buffer gas at a pressure of 1-3 x 10" 4 Torr. 
Several different fragment ions were observed and are reported in Table 4. 

Ekern et al. m extended their earlier study by examining the photostability of a 
series of twenty-four PAH cations and one fullerene, C 60 + . Electron impact and laser 
desorption were used to generate the ions which were then trapped and mass-analyzed by 
an FT-ICR mass spectrometer incorporating a 3 T magnet. The trapped ions were 
subjected to irradiation from a xenon arc lamp and the fragmentation products were 
recorded. From this series of PAH cations, it was discovered that the observed fragmen- 
tation patterns fell into one of four categories: photostable, loss of hydrogen atoms only, 



78 



Table 4. Observed fragmentation channels and efficiencies for selected PAH cations 
using the ion-trap detector. (Reproduced from reference 197) 



_ b £X%) 





(M-Hr 


(M-2HT 


(M-3Hr 


(M-4HT <M-2C.2HT(M-2C.3Hr<M-2C4HT(M-4C.2HT 




naphthalene 


100 


95 





10 








1 


90 


acenaphthylene 


100 


60 





20 











40 


acenaphthene 


100 


30 





5 











75 


phenanthrene 


5 


100 





10 35 


15 


20 





75 


anthracene 


100 


65 





10 50 


20 


30 





65 


pyrene 


40 


100 


20 


60 











65 


coronene 


35 


100 





25 


10 


15 





75 


corannulene 


40 


100 


10 


100 











50 



a The relative uncertainty in the relative abundance is about fifteen percent. 

b The fragmentation efficiency, £ ft is the fraction of the product ion intensity following CID expressed as a 
percentage. It is the ratio of the sum of the daughter ion intensities to the total ion intensity of the daughter 
ions and the undissociated parent ion detected after CID. 



loss of hydrogen and carbon atoms, and photodestroyed. Their results are condensed in 
Table 5. No real correlation was found to explain why certain PAH cations fragment one 
way while a very similar cation will fragment in a different fashion (e.g. naphthalene vs. 
anthracene). 

Current Efforts 
Our efforts to study the photodissociation of PAHs of astronomical importance 
began where the work of Ekern et al. ,98 left off. Our initial plans were to continue with 
their work by examining wavelength-dependent photodissociation of the same ions. In 
the previous work, each of the twenty- four ions was generated, trapped, and irradiated 
with the full spectrum from a xenon arc lamp for 500 ms. After the 500 ms irradiation 



79 



Table 5. A list of the twenty-four PAHs examined by Ekern et al. placed within the 
appropriate fragmentation category. 



Photostable 

acenaphthylene 
azulene 
biphenylene 
fullerene, C 60 



Loss of H atom(s) 



fluoranthene 

pyrene 

perylene 

benzo[g/i/]perylene 

acenaphthene 

coronene 

triphenylene 

fluorene 



T ,oss of H and C atom(sl Photodestroved 

naphthalene 



phenanthrene 
chrysene 
anthracene 
diphenylacetylene 
benz[a] anthracene 
benzo [k] fluoranthene 
benzo [a] pyrene 
tetracene 



decacyclene 



naphtho[2.3-a]pvrene dibenzanthracene 



period the resulting mass spectrum was recorded. We believed a closer study of the 
dissociation was warranted for these systems. We generated ions with internal EI and 
trapped them in an open-ended analyzer cell. The trapped ions were mass analyzed by a 
FT-ICR mass spectrometer incorporating a 2 T magnet. Several modifications were 
made, including the design and construction of a much larger cell to allow more light to 
enter. A computer-controlled mechanical shutter was also built in-house. The source of 
irradiation was an argon ion laser used to pump a dye laser. After several months of 
designing, constructing, and troubleshooting, the studies were ready to begin. At first, we 
did not observe any photodissociation for the PAHs of interest. We then decided to work 
with a different molecule that would easily dissociate, ;?-bromochlorobenzene. After 
some success with this system and after becoming familiar with the experimental setup, 
we switched back to the PAHs. Once again, we had no success. It was decided that we 
simply were not accessing the appropriate wavelengths for photodissociation with our 



80 
setup. Thus, we decided to take a step back, and use the same xenon arc lamp that Ekern 
et al. m used. Our thought was to repeat the experiments while using in-line filters so that 
we could narrow down the appropriate wavelengths in order to purchase the correct laser 
dyes. While performing the filter studies, I decided to change the experiment slightly 
after reading the paper published by Boissel and co-workers. Instead of opening the 
shutter each time for 500 ms, I started to extend this time. The results of this variation 
were (as Boissel had witnessed) that at longer irradiation times (up to 60 s) the mass 
spectra drastically changed from what Ekern et al m had reported. Since that time, the 
photodissociation of the PAHs has been studied by varying the length of irradiation from 
the xenon arc lamp. The next two chapters will report the findings for the fluorene cation 
as well as the naphthalene, acenaphthylene, and the diphenylacetylene cations. 



CHAPTER 4 

PHOTODISSOCIATION AND ION-MOLECULE 

REACTIONS OF FLUORENE CATIONS 

Introduction 

Photodissociation and ion-molecule reactions of fluorene cations using Fourier 
transform ion cyclotron resonance mass spectrometry are discussed in this chapter. First, 
a brief background of relevant theoretical work will be presented. Next, a review of the 
experimental setup used to study fluorene will be offered, including photodissociation 
studies using a xenon arc lamp and ion-molecule reactions without the lamp. A mention 
of plausible structures for some of the products formed from both lamp-on and lamp-off 
experiments will be provided. The chapter will end with concluding remarks and a 
mention of future experiments that need to be carried out. 

Background 

Fluorene (C 13 H 10 , see Figure 33) falls into the category of nonalternant polyarenes. 
A nonalternant polyarene is defined as a PAH molecule that comprises one or more rings 
that are other than fused six-membered benzenoid rings. 199 

Previous work performed 198 on the fluorene cation revealed that upon irradiation, 
up to five hydrogen atoms were lost from the parent ion. Density functional theory 
calculations have been carried out at the B3LYP/4-3 1G level of theory to determine the 
most likely positions for the hydrogen atom losses. 200 The results of the calculations 



81 



82 




Figure 33. Chemical structure and numbering system of the fiuorene molecule (hydrogen 
atoms have been omitted from the structure). 



reveal that the first hydrogen loss is from the sp 3 carbon atom at the number nine position. 
The remaining four hydrogen atoms are lost in a sequential order around one of the 
aromatic rings. These calculations were employed to determine the energies of the 
appropriate cations which might be formed in the photodissociation experiment with no 
consideration given to the potential energy barriers that exist on the pathway from one 
cation to the next. Figure 34 depicts a flow diagram of the energy pathways leading from 
the parent ion at a m/z of 166 to the daughter ion at m/z 161. 

Experimental 
The underlying principles of FT-ICR MS have been presented earlier. Mass 
spectra were acquired on a home-built FT-ICR mass spectrometer incorporating an 
IonSpec data station (IonSpec, Corp., Irvine, CA) along with a 2 Tesla superconducting 
magnet (depicted in Figure 35). The analyzer cell used throughout these studies was a 
cylindrical cell built in-house (see Figures 36-38). Fiuorene samples were placed on the 
end of a solids probe and introduced directly into the vacuum chamber through a small 
gate valve located on the vacuum chamber. The vacuum port which supported the probe 



83 




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84 
assembly was pumped out by a mechanical pump (Alcatel Vacuum Products, Hingham, 
MA). Operating pressures in the analyzer cell were typically between 4-6 * 10' 8 Ton- 
maintained by a 700 L s" 1 oil diffusion pump (Alcatel Vacuum Products, Hingham, MA) 
that was backed by a second mechanical pump. 

The analyzer cell was held in position by four stainless steel rods that were 
connected to a flange at the end of the vacuum chamber; this flange also contained all of 
the electrical feedthroughs. Due to the weight of the entire cell assembly (rods, rings, EI 
gun, analyzer cell, connections, etc.), a stainless steel set screw was used to support the 
free end of the cell assembly. Effort was put forth to ensure that the center of the analyzer 
cell was located as near as possible to the center of the magnet. 

A typical pulse sequence used to generate the mass spectra is depicted in Figure 
39. The quench pulse simply emptied the analyzer cell of any unwanted ions that were 
left over from a previous experiment. In these experiments the quench pulse was set for 
an unusually long time (500 ms). The extra time was necessary to ensure that the 
mechanical shutter had enough time to actually come to a closed position. Normally, the 
quench pulse would be set to ca. 5-10 ms. After the quench pulse there was a short delay 
period; a similar delay (3 ms) was placed between each event to allow the electronics 
(particularly the reed relays) to "settle" before the next event. Ions were generated by 
electron impact ionization of the neutral molecules during the next pulse (usually between 
20-50 ms). Electrons were emitted from a heated rhenium filament and accelerated by a 
potential set by the difference between the ionizing voltage (usually set only a few eV 
above the ionization potential to minimize the amount of fragmentation from the 



85 



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mass spectrum for the photodissociation studies of the fluorene cation. 



ionization process) and the trapping tubes. Even with careful setting of the electron 
energy, some fragmentation of the parent ion occurred as a result of the EI process; 
therefore, the next event in the pulse sequence involved ion isolation. This was 
accomplished by placing an RF sweep over the mass range of unwanted ions or by 
placing an RF burst directly on an unwanted ion and driving it out of the analyzer cell. 
Following isolation was the irradiation event, initiated by a computer-generated TTL 
pulse, that signaled the opening of the mechanical shutter for the specified amount of 
time. In order to detect the presence of any ions after irradiation, the ions were excited to 
larger cyclotron orbits to generate the image current that was measured on the detection 
plates. This image current was amplified, digitized, and stored for processing by the 
computer. 

Studies pertaining to the fluorene cation involved two main themes; detecting 
photodissociation products and/or detecting ion-molecule products. Photodissociation of 
the trapped parent ions was accomplished by irradiation supplied by an LX300UV xenon 
arc lamp (ILC Technology, Sunnyvale, CA) that emitted wavelengths ranging from about 



90 
200 to 1 100 nm. The lamp was aligned so that the maximum amount of light was 
transmitted through a window on the end of the vacuum chamber and allowed to enter 
into the analyzer cell due to the open-ended nature of the cell. The lamp was positioned 
about a meter from the analyzer cell to minimize effects on it from the fringe field of the 
magnet; therefore, a focusing lens was employed to narrow the beam into the analyzer 
cell. A computer-controlled mechanical shutter was used to allow the light into the 
analyzer cell. This was accomplished by the addition of a user pulse within the pulse 
sequence which has been discussed previously. At long irradiation times, ion-molecule 
reactions compete with the photodissociation process. Ion-molecule reactions also occur 
with longer delay times that are placed before the detection event (lamp-off experiments). 
During both of these types of experiments (long irradiation times and long delay times) 
larger molecular weight ions began to appear in the mass spectra. These ions will be 
discussed below. 

Results and Discussion 
Photodissociation vs. Irradiation Time 

As a result of irradiation from the full spectrum of a 300 W xenon arc lamp, the 
fluorene cation loses up to five hydrogen atoms, depending on the irradiation time. The 
variable in this set of experiments was the length of the irradiation pulse. The pulse 
sequence used for these experiments is reproduced in Figure 40. Figures 41 and 42 chart 
the percent abundance of ions between m/z 166 and 161 as a function of the irradiation 
time along with typical mass spectra (Figures 43- 45) representing three different 
irradiation times. 



OTHER PARAM, 

PAH: Fluorene 
File: DK981210.A01 

m/z 167 
Using BNC 15 


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The first daughter ion (m/z 165) is observed in the mass spectrum at t = ms. 
The ion isolation pulse was found not to be 100% effective at isolating the parent ion at 
m/z 166, as demonstrated by the abundance of the ion at m/z 165 which resulted from the 
ionization process. The parent ion had practically disappeared by the time the irradiation 
approached 300 ms, suggesting that the first hydrogen atom is lost quite easily. Along 
with this decrease in the parent ion, there was a sharp increase in the abundance of 
[M-H] + (where M represents the parent molecule). As mentioned previously, theoretical 
studies predicted that this hydrogen atom was lost from the sp 3 carbon atom located on 
the five-membered ring. The abundance of [M-H] + increased rapidly, reaching a 
maximum near 200 ms where it began to decrease due to the loss of a second hydrogen 
atom, resulting in [M-2H] + . After irradiation for 2000 ms, practically all of [M-H] + was 
consumed to produce [M-2H] + . 

The abundance of [M-2H] + never reached a level comparable to ions from which 
it was derived; this indicates that [M-2H] + is possibly not a stable structure, and therefore, 
allowed for further dissociation. This ion began to increase after irradiation of about 100 
ms and reached a maximum at about 400 ms. This ion, like [M-H]\ is practically gone at 
long irradiation times. The loss of a third hydrogen atom resulted in the next ion, 
[M-3H] + , which first appeared after approximately 200 ms of irradiation. The abundance 
of this ion sharply rose to a maximum at about 1000 ms, where it essentially remained 
constant out to 5000 ms. A possible explanation as to why the abundance of [M-3H] + 
does not rise to a maximum and decrease like the previous daughter ions could be that 
[M-3H]" is being formed faster than it is being consumed. There are at least two possible 



98 



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mechanisms that could account for the consumption of the [M-3H] + . First, [M-3H] + 

could simply continue to dissociate to form the next daughter ion, [M-4H] + . The second 

possibility is that [M-3H] + may be involved with ion-molecule reactions that result in ions 

of larger molecular weight. An example of these ions is shown in Figure 46. These ions 

are of importance and interest and will be discussed in a later section. 

The next ion in the series is [M-4H] + , which slowly began to increase at about 400 
ms. The abundance of this ion, like [M-2H]\ increased to a maximum and then 
decreased as the final daughter ion began to appear. The last ion, [M-5H]\ appeared to 
behave in a similar fashion to [M-3H] + . It was the last ion to appear (after about 500 ms 
of irradiation), and its abundance continued to increase out to an irradiation time of 5000 
ms. Again, the production of [M-5H] + (presumably from the [M-4H] + ) occurs at a faster 
rate than its consumption. In this case, the consumption of the [M-5H] + is solely 
attributed to ion-molecule reactions, since the loss of more than five hydrogen atoms has 
not been observed with the experimental design of any of the current or previous 
experiments 198 involving the fluorene cation. 
Atomic vs. Molecular Hydrogen Loss 

The question of whether the loss in mass of five amu from the parent ion is by 
sequential hydrogen atom loss, or possibly, by the loss of a hydrogen molecule was 
addressed. After ionization of the parent molecule, an isolation pulse was added to 
isolate the parent ion. The experiment used an irradiation time of 1000 ms which was 
kept constant. To address the aforementioned question, the experiment was conducted 
twice. The first experiment simply recorded the mass spectrum of the fluorene ion with 





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104 
an irradiation time of 1000 ms (see Figure 47). In the second experiment, an ejection 

pulse was introduced into the pulse sequence (see Figure 48). In the first set of 
experiments (with no ejection pulse), the mass spectrum should be dominated by [M-3H] + 
(as well as smaller amounts of [M-4H] + and [M-5H] + ). In the second experiments (with 
the ejection pulse turned on), [M-3H] + should be absent from the mass spectrum (as well 
as [M-4H] + and [M-5H] + ). Figures 49 and 50 are the mass spectra for the experiments 
with no ejection pulse and the ejection pulse turned on, respectively. From Figure 49, it 
is clear that [M-3H] + is the dominant ion at an irradiation time of 1000 ms, along with 
smaller contributions from the other two ions. On the other hand, Figure 50 shows a very 
clean mass spectrum with [M-3H] + , [M-4H] + , and [M-5H] + being absent from the 
spectrum. This clearly demonstrates that at least for one step of the process, the photo- 
dissociation of the parent ion is due to the loss of a hydrogen atom and not by the loss of 
a hydrogen molecule. The experiments were repeated with the ejection pulse placed on 
the ions at m/z 165, 163, and 162 with similar results; hydrogen atoms were lost, not 
hydrogen molecules. 
Ion-Molecule Reactions 

As mentioned earlier, at long irradiation times, products of ion-molecule reactions 
begin to appear and they are ions with larger molecular weights. A typical mass spectrum 
of this situation is depicted in Figure 51, where the fluorene cation was irradiated for 
4000 ms. After irradiation for 4000 ms, the [M-3H] + is the primary fragment ion 
remaining, while the parent ion at m/z 166 is essentially gone. However, with the 
irradiation time extended to much longer times, ion-molecule reactions began to generate 












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108 
an ion at m/z 324, which may undergo photodissociation to form smaller fragment ions. 

If the lamp is turned off for this 4000 ms, the higher molecular weight ions are absent 
from the mass spectrum, as shown by Figure 52. Here, the conditions are exactly the 
same except the shutter was not allowed to open. To prove that the ion at m/z 324 did not 
form at shorter irradiation times, the experiment was again repeated with identical 
conditions except the irradiation time was only 500 ms, the results are depicted in Figure 
53. 

A possible explanation for the formation of the ion at m/z 324 comes as the result 
of an ion-molecule reaction involving the neutral background parent fluorene molecules, 
which were always present at a pressure of approximately 5 x 10" 8 Torr, along with a 
daughter ion from the fluorene cation. The Z>/-9H-fluoren-9-ylidene (bifluorenylidene) is 
a likely structure as the initial product from the reaction (see Figure 54a). If this ion is 
subjected to sufficient irradiation it is possible for a number of hydrogen atoms to be 
stripped away resulting in the dimdex\o[\,2,3,4-defg;\',2\y,4'-mnop]chrysem ion at m/z 
324 (see Figure 54b). Bifluorenylidene is a known molecule that has been characterized 
in a number of ways 201,202 including by x-ray crystallography. 203 The structure in Figure 
54b is also a known molecule that has been isolated and studied. 202 The proposed 
structure for the ion that is formed at m/z 324 is a piece of a "Buckyball" known as a 
"Buckybowl". If this is truly the structure for this ion, might longer irradiation times 
(minutes) result in the formation of fullerenes of different sizes? If this were proven true, 
a new wave of interest in studying PAH cations using FT-ICR MS would prevail with 



109 




7\" 




m/z 328 



bi-9H-fluoren-9-ylidene 
(bifluorenylidene) 



(a) 



hv 




m/z 324 



diindeno[ 1 ,23A-defg: 1 ',2',3',4'-mnop]chrysene 
("Buckybowl") 



(b) 



Figure 54. Structure (a) results from a reaction between the neutral fluorene molecules 
and the fluorene fragment ions. The resulting ion of m/z 328 can further lose hydrogen 
atoms to form (b) which has a m/z of 324. 



collaborations between the astrophysical community and chemists. At the present time, 
no effort has been exerted to isolate and photodissociate the ion at m/z 324. 



CHAPTER 5 

PHOTODISSOCIATION AND ION-MOLECULE REACTIONS 

OF ACENAPHTHYLENE, DIPHENYLACETYLENE, 

AND NAPHTHALENE CATIONS 

Introduction 

The previous chapter concentrated solely on the photodissociation and ion- 
molecule reactions of the fiuorene cation. The present chapter extends these studies to 
include cations from the three remaining photodissociation groups (as defined by Ekern et 
al. m ). Specifically, the chapter will be broken down into three sections involving the 
acenaphthylene cation, the diphenylacetylene cation, and the naphthalene cation. These 
three cations were photostable, lost hydrogen and carbon atoms, and were photo- 
destroyed, respectively, after a 500 ms irradiation pulse from a xenon arc lamp. 198 A 
discussion regarding possible structures for the observed products will be offered along 
with remarks on future studies. 

Acenaphthylene 

Acenaphthylene (C 12 H g , see Figure 55) is a member of the largest category of 
nonalternant polyarenes which contain one or more five-membered rings. Previous work 
on the acenaphthylene cation indicated that this cation was photostable after irradiation 
for 500 ms by a xenon arc lamp. 198 Since the acenaphthylene cation was photostable 
under the conditions of the earlier experiments, there was no effort to perform any of the 



110 





















Ill 




Figure 55. Chemical structure and numbering system for the acenaphthylene molecule 
(the hydrogen atoms have been omitted from the structure). 



theoretical calculations on fragment ion structures as were previously presented for the 
fluorene cation. 

The experimental setup and procedures were the same as previously described so 
no details will be given. A series of experiments were carried out using very long delay 
times (with the lamp turned off). This delay was simply placed in the pulse sequence 
immediately before the detection event. At short delay times, no significant changes in 
the mass spectrum were detected; the spectrum is dominated by the parent ion at m/z 152 
(see Figure 56). But as the delay time increased, a definite change was observed in the 
mass spectrum. Figure 57 affirms the formation of a new ion near m/z 304 which 
resulted from an ion-molecule reaction between a neutral acenaphthylene and a 
acenaphthylene cation. The structure of the 304 ion is most likely that of the known cis 
or trans cyclobutane-like dimer 204 " 209 (see Figure 58). It should be possible to design an 
experiment to check this assumption. After the long delay time that is necessary to 
generate the ion, the ion at m/z 304 could be isolated and then subjected to irradiation 



112 



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115 
from the xenon arc lamp. The resulting photodissociation mass spectrum could then be 

compared to the photodissociation mass spectrum obtained from a purchased or 

synthesized sample. 

The photostability of the acenaphthylene cation that was previously reported was 
confirmed. However, when longer irradiation times were used, a new ion began to appear 
in the mass spectrum at m/z 228 (see Figure 59). Since this ion was not observed in the 
previously discussed experiment (with the lamp off), it is assumed that the ion is a 
product from the photodissociation of a larger ion; in this case the cis or trans 
cyclobutane-like dimer. The assumption is that this ion resulted from the photodissoc- 
iation of the previously mentioned ion at m/z 304. The ion at m/z 228 arises from a loss 
of 76 from the dimer-like ion. The proposed scheme for the generation of this ion is 
depicted in Figure 60. It is possible that this ion undergoes a rapid rearrangement to form 
the last structure shown in Figure 60. As mentioned before, this proposed structure could 
be proven if the compound was commercially available. 

Diphenylacetylene 

Diphenylacetylene is not a true PAH per se, but nonetheless it was a molecule that 
was previously studied. 198 Figure 61 depicts the structure of the diphenylacetylene 
molecule. The experimental procedures used to study the diphenylacetylene cation were 
the same as those used for the previous systems. Diphenylacetylene cations were 
generated by internal electron impact and both photodissociation (lamp on) and ion- 
molecule (lamp off) products were mass analyzed using FT-ICR MS. 












116 




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The first set of experiments involved placing long delay times before the detection event 
(lamp off experiments). Figure 62 depicts a characteristic mass spectrum of the diphenyl- 
acetylene cation after a delay time of only 500 ms. The mass spectrum is dominated by the 
parent ion at m/z 178 along with traces of ions at m/z 152 and 356. The mass spectrum 
drastically changed when the delay time was extended. Figure 63 is the mass spectrum following 
a delay time of 5000 ms. Following this extended delay time, the ion at m/z 356 dominated the 
mass spectrum. The abundance of this ion increased as a function of delay time as illustrated in 
Figure 64. Along with the increase in the ion at m/z 356, there was an equal decrease in the 
parent ion at m/z 178 indicating that the parent ion is directly related to the formation of the ion 
at m/z 356. In fact, the product ion (m/z 356) most likely results from an ion-molecule reaction 
occurring between a neutral parent and a parent ion to form a "dimer-like" species. Two possible 
structures could account for the ion at m/z 356. The first possibility is that a neutral 
diphenylacetylene and a diphenylacetylene cation simply come together and stack on top of one 
another (see Figure 65). The second possibility involves the formation of bonds to create a 
"pinwheel" molecule with a four-membered ring in the center (see Figure 65). 



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Figure 65. Two possible structures for the ion at m/z 356 that results from an ion- 
molecule reaction involving a neutral diphenylacetylene and a diphenylacetylene cation. 



An experiment was devised to ascertain which of the two structures (assuming 
one of these two is the correct structure) was the correct one. The plan was to isolate the 
ion at m/z 356, and then irradiate the ion with the xenon arc lamp. The idea was that if 
the stacked structure was the correct assumption, it should dissociate relatively easily 
(after a short irradiation pulse) since this structure is held together through weak 
interactions. If the pinwheel structure is the true structure, it is assumed that a much 
longer irradiation pulse would be necessary to dissociate the ion. Also, upon 
photodissociation, the pinwheel structure would most likely result in several product ions, 
not just simply reversing back to the parent ion at m/z 178. Unfortunately, these 
experiments have not been attempted as of this time, but hopefully, they will be 
implemented in the near future. 



123 
The second set of experiments performed on the diphenylacetylene cation 
analyzed the photodissociation products as a function of irradiation time. With long 
irradiation times, ion-molecule reactions begin to compete with photodissociation 
processes. Figure 66 depicts the mass spectrum of the diphenylacetylene cation after 
irradiation of 500 ms. Comparison of this spectrum with the mass spectrum in Figure 62 
shows an increase in the abundance of the ion at m/z 152. The assumption is that the 152 
ion is formed from a diphenylacetylene cation that has lost an acetylene group, which 
would agree with the photodissociation observed by Ekern et al. m When the irradiation 
pulse was extended, several new ions began to appear in the mass spectrum (Figure 67). 
Plausible structures have not been assigned to these new peaks as of yet. They are most 
likely different photofragments derived from the previously mentioned ion at m/z 356. 
As previously stated, the ion at m/z 356 is generated after long delay times, but in this 
case, as soon as the ion is formed it subsequently photodissociated into a number of 
smaller fragment ions. 

From the experiments just described, it is clear that ion-molecule reactions are 
occurring when long delay times before the detection event are employed. More 
experiments need to be performed in order to postulate on the structure of the ion at m/z 
356. It is also quite evident that long irradiation times generate larger ions that are 
derived from the ion at m/z 356. Again, more work is needed before any structures can 
be assigned to these ions. 



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Naphthalene 

The last PAH cation to be discussed in this dissertation is that formed from 
naphthalene. Naphthalene is the simplest member of the nonalternant polyarene group. 
Previous work on the naphthalene system determined that the naphthalene cation was 
photodestroyed 198 after only 500 ms of irradiation from a xenon arc lamp. The two 
proposed routes for the photodestruction of the naphthalene cation are given in Figure 68. 

The experimental parameters used for the naphthalene system were, again, the 
same as with the previous studies. Ion-molecule reactions, resulting from long delay 
times, were not investigated for the naphthalene cation. Instead, the experiments 
concentrated on the photodissociation of the cation. Figure 69 depicts a typical mass 
spectrum of the naphthalene cation after electron ionization. The mass spectrum shows 
small amounts of dissociation of the cation from the electron impact process itself. A 
contrasting view is seen in Figure 70 where the naphthalene cation was subjected to an 
irradiation of 5000 ms from the xenon arc lamp. Again, there is a large abundance of the 
ions at m/z 102 and 76 (the first two photofragments leading to the total destruction of the 
ion). But, the spectrum also depicts several new ions, most notably at m/z 202 and 250. 
Figure 72 offers possible structures for the 202 and 250 ion. The first scheme depicts a 
neutral naphthalene and a naphthalene cation forming new bonds to generate the benzo- 
[g/z/]perylene cation. The second scheme shows the photodissociation of the benzo- 
[g/?/]perylene cation followed by a rearrangement resulting in the formation of the 
fluoranthene cation. Both of these structures are reasonable and should be relatively 
straightforward to prove. 



127 



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reference 198) 



A set of experiments were devised and attempted that would hopefully elucidate 
the structures of the ions at m/z 202 and 250. Benzo[g/z/]perylene and fluoranthene are 
two systems that were previously studied and are readily at hand. The idea was to fashion 
an experiment where the naphthalene cation would be irradiated for 5000 ms, then the ion 
of choice (m/z 202 or 250) would be isolated using a simple isolation pulse, and finally 
the selected ion would be subjected to another irradiation pulse lasting 500 ms (to match 
the conditions used by Ekern et al. I98 ). Ultimately, the resulting mass spectrum would be 
compared to the previous work for a match of photodissociation products (loss of 



128 



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hydrogen atoms only for both cations). These experiments were attempted several times 

with little success. The results were encouraging enough to continue once a few technical 
problems were overcome. At first, attempts to open the shutter twice within the same 
pulse sequence did not work. After a little assistance from the electronics shop, this 
problem was defeated. The next major hurdle is to decrease the length of the irradiation 
pulse in order to keep as many ions in the analyzer cell as possible, since the longer the 
amount of time before detection of the ions, the more the ion population decreases. But 
at the same time, the irradiation pulse has to be long enough to provide an adequate 
number of ions in order to produce a mass spectrum. It is this author's belief that the 
above mentioned experiments will be successfully completed. 

A very brief remark pertaining to the output of the lamp and the proposed 
dissociation mechanisms (for example, Figure 68) is given below. The naphthalene 
cation has several very strong absorption features in the uv region (308 nm for example). 
The output from the LX300UV lamp is between 200-1000 nm. A quick calculation of 
the amount of energy needed to rupture the naphthalene cation (as shown in Figure 68) 
shows that roughly 388 KJ mol" 1 is required to break the indicated bonds in the proposed 
mechanisms. The output from the lamp provides several eV of energy (perhaps three or 
four eV of energy) per photon. It is reasonable to suggest that these ions can absorb two 
or more photons (especially considering the amount of time they are trapped in the 
analyzer cell) and therefore acquire more than enough energy to fragment in the manner 
as proposed in this dissertation. More detailed experiments will, of course, be necessary 
in the future to ascertain the validity of such claims. 



CHAPTER 6 
CONCLUDING REMARKS 



Binding Energies for CD:Amino Acid Complexes 
As mentioned earlier, a great deal of information cannot be inferred from these 
data due to the limited number of systems. For this reason, there are limited conclusions 
that can be drawn from the available data. Threshold binding energies of 1 .32 and 
0.58 eV were determined for the complexation of tryptophan to a-CD and P-CD, 
respectively. Without further examination of these two systems, the absolute numbers 
may not be accurate, but the relative numbers do seem to be believable. Since the cavity 
of the a-CD is the smaller of the two systems, it makes sense that this host would bind 
more tightly to its guest than the p-CD host would bind to the same guest. 

The second conclusion that can be inferred from these data pertains to the 
threshold binding energy determined for a-CD incorporating a lysine, tryptophan, or 
proline guest, since the cavity size in each case remains the same. Threshold binding 
energies of 1.32, 1.21, and 0.71 eV were determined for the a-CD binding with 
tryptophan, proline, and lysine guests, respectively. Again, the relative numbers are 
reasonable for this series. The binding strength should be the greatest for tryptophan and 
the weakest for lysine (side chain is a simple straight chain and would posses the weakest 
intermolecular forces). As for the tryptophan and proline case, they both possess a ring 

132 



133 

but the tryptophan would extend deeper into the cavity. In addition, the intermolecular 
forces holding the tryptophan complex together would be stronger due to the increased 
mass over the proline complex (all other intermolecular forces taken to be equal). 

Much more work can and should be done with these systems in the near future in 
order to draw better conclusions. Similar work in other laboratories is underway to study 
the binding of small peptides and proteins to substituted CDs (more water soluble). The 
CID experiments performed here were not terribly successful for a number of reasons. 
Most of the work was performed using argon as the collision gas. Only at the end of the 
project did krypton gas become available. Work in the future could reasonably benefit 
from an even heavier (but more expensive) gas such as xenon. Many of the leading 
scientists in this field agree that xenon gas is the best choice for a collision gas. A 
different method for measuring binding energies in the gas-phase may be beneficial to 
this project. Due to the size of the CDs (many degrees of freedom), the binding energies 
may be obtained more easily using a relatively new technique known as BIRD 
(Blackbody Infrared Radiation Dissociation). Basically, the complexes are trapped in the 
analyzer cell and allowed to dissociate by absorbing photons that are emitted from the 
heating of the entire analyzer cell. An initial design incorporating a heated tube to slide 
over the analyzer cell has been fabricated and tested with little success. Hopefully, this 
project will continue, but that will be left up to a future student to decide. 

Fluorene 

The fluorene cation has received more attention than any of the other PAH cations 
presented in this work. The most obvious observation that can be made from the 



134 
presented data, is that the processes that are occurring are very complicated. What 
complicates the studies is that the lamp is driving the parent ion at m/z 1 66 down to 
daughter ions at m/z 165, 164, 163, 162, and 161. If long delay times are used (no lamp) 
each of these ions is capable of undergoing an ion-molecule reaction with a neutral parent 
to generate an ion between m/z 331-327. But, when the lamp is used, each of these ions 
(m/z 331-327) can then fragment, resulting in smaller ions. 

From the experiments that measured the photodissociation as a function of 
irradiation time, one can monitor the increase and decrease in the abundance of the 
daughter ions at m/z 165, 164, and 162. The abundance of the daughter ions at m/z 163 
and 161 did not decrease with increasing irradiation time. The idea here is that these two 
ions are forming faster than they are being consumed (presumably through ion-molecule 
reactions). The loss in mass of five amu from the parent ion was also shown to occur by 
sequential hydrogen atom loss and not by the loss of hydrogen molecules. Experiments 
utilizing long delay times were presented and demonstrated the formation of new ions 
that resulted from ion-molecule reactions. These ions were subsequently 
photodissociated after the application of long irradiation times. A possible structure for 
the ion at m/z 324 was presented, but more experiments need to be conducted in order to 
prove (or disprove) the validity of this structure. With the completion of these 
experiments, I hope there is a little better understanding of the photodissociation 
processes as well as ion-molecule reactions that are occurring with fluorene cation. 



135 
Acenaphthylene 

The acenaphthylene cation was photostable after irradiation for 500 ms (as was 
previously determined 198 ). But, with long delay times, ion-molecule reactions did occur 
that generated an ion at m/z 304. The structure of this ion is most likely that of the cis or 
trans cyclobutane-like dimer. 204 " 209 Irradiation of this ions resulted in the formation of an 
ion at m/z 228. A proposed structure for this ion (after a rearrangement) was given. A 
new set of experiments will need to be conducted to prove the validity of this structure. 

Diphenylacetylene 

Ion-molecule reactions were also observed at long delay times for the 
diphenylacetylene cation as evident by the formation of a new ion at m/z 356. Two 
structures were proposed for this ion, along with a relatively straightforward experiment 
that should be able to determine which structure is the correct one. Irradiation of the 
parent ion (m/z 178) produced an ion at m/z 152 which is assumed to be the loss of an 
acetylene group. This observation is in agreement with Ekern et al. I98 Longer irradiation 
times resulted in the generation of several new ions ranging from m/z 276-354. To date, 
no structures have been proposed for any of these ions. 

Naphthalene 

There were no lamp off (long delay times) experiments performed on the 
naphthalene cation. After short irradiation times, the naphthalene cation was found to be 
photodestroyed (as was previously reported 198 ). But when the irradiation time was 
extended, the mass spectrum depicted the formation of several new ions. Two of the 
more abundant ions were observed at m/z 202 and 250. Structures were proposed for 



136 
both of these ions. Again, a set of experiments were described that should justify the 
proposed structures. 

As has been mentioned previously, much more work needs to be carried out on 
the four cations described in this thesis. In addition to these cations, there are still the 
remaining twenty from the previous study 198 that can be examined. It is with deep regret 
that this author got involved with this project so late in his career. The amount of work 
that is possible within this area is massive and should keep several students busy for the 
next few years. Lamp off (ion-molecule reactions) and lamp on (photodissociation) 
experiments could be performed on hundreds of PAH cations. Eventually, after 
significant new information has been retrieved for these systems, one could go back to the 
original starting point of this project, which was wavelength-dependent studies using the 
argon ion laser. The Eyler laboratory is equipped with a Nd:YAG dye-pumped laser and 
has access to an argon ion laser. The possibilities are endless. Along with the available 
equipment (lasers and three FT-ICR mass spectrometers), the Eyler laboratory has just 
upgraded the data station on the 2 T instrument. The instrument is now controlled by the 
MIDAS data station which should allow for more sophisticated pulse sequences which 
include much improved trapping, isolation, and ejection of ions. With the capability of 
designing more sophisticated pulse sequences, many of the described experiments that 
could prove proposed structures, should be possible. 












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BIOGRAPHICAL SKETCH 

David Kage was born on June 15, 1969 in Galesburg, Illinois. He grew up in East 
Galesburg, where he attended grade school. He then attended middle and high school in 
Knoxville, Illinois, graduating in 1987. High school was where he became interested in 
science thanks to Mr. Ron Zarn, his teacher for algebra, geometry, trigonometry, calculus, 
physics I and II, and of course chemistry I and II. 

During the next six years, he attended Illinois State University in Normal, Illinois, 
where he received his B.S. degree in chemistry in May of 1991 and his M.S. degree in 
chemistry in August of 1993. His thesis, "The Effects Of Extra Neutrons In The Carbon 
And Hydrogen Framework Upon The Thermodynamics Of Intermolecular Electron 
Transfer," involved isotope enrichments utilizing the differences in solution electron 
affinities between aromatic hydrocarbons and their isotopically substituted analogues. 

He immediately began his studies at the University of Florida in the fall of 1993. 
While attending the University of Florida, he decided to try a different area of 
experimental physical chemistry, so he joined the research laboratory of Dr. John Eyler. 
His current research interests are photodissociation studies of gas-phase polycyclic 
aromatic hydrocarbons of interstellar importance utilizing Fourier transform ion cyclotron 
resonance mass spectrometry. 



150 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




(I £ 



R. Eyler, Chairm 
ssor of Chemisti 




I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 



■ L ■ ^ 



William Weltner 
Professor of Chemistry 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 





^Ma 



Cobert J. Hanra 
Professor of Chemistry 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosopr 




~7 

Lisa McElwee-White 

Professor of Chemistry 






I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Laszlo Prokai 

Associate Professor of Pharmaceutics 



This dissertation was submitted to the Graduate Faculty of the Department of 
Chemistry in the College of Liberal Arts and Sciences and to the Graduate School and 
was accepted as partial fulfillment for the degree of Doctor of Philosophy. 

December. 1999 



Dean, Graduate School 









LD 

176 

199£ 



UNIVERSITY OF FLORIDA 



3 1262 08554 8435